Chapter 9/10
What are the most abundant phospholipids in animal cells?
- 3 carbon glycerol backbone esterified to 2 fatty acid chains -other carbon in glycerol attached to phosphate -phosphate attaches to different head groups
What is phase contrast microscopy?
- generate image where degree of brightness depends on refractive index of cell -can visualize cells without fixation as fixation can destroy some cells - see thin layers of cells, but not thick tissues (thick part retards light and phase shifts relative to light that has passed through adjacent thinner region) - examine location and movement of large organelles -exploit interference effects when 2 rays of light combine to create an image of cell structure (1 pass through thick, 1 through thin)
How many species in the lipid bilayer?
-500-1000 different species make up -also have structural lipids: inositol phospholipid involved in cellular communication and can form docking sites and recruit other proteins
What is a light microscope?
-A microscope that uses a series of glass lenses and visible light to magnify an image -This microscope magnifies images up to 1,000 times the actual size. -light waves travel using optical diffraction -light has wavelike nature, not in straight line
What are channels in the membrane made up of?
-B barrels form large channels -crystallize quickly into rigid structures -can create pores to secrete molecules -in outer membrane of mitochondria and chloroplast ex: porin: 16 B sheets and ions move in/out -smaller barrels with AA side chains in middle function as receptors and anchors for proteins to bind orient proteins
What is Immunogold electron microscopy?
-Electron microscopy technique in which cellular structures or molecules of interest are labeled with antibodies tagged with electron-dense gold particles, which show up as black spots on the image. -can incubate thin section of cell with primary antibody -2nd antibody with colloidal gold particle attaches -gold particle electron dense and seen as black dot on microscope
What is FRAP and how does it work?
-Fluorescence Recovery After Photobleaching -Used to test protein motility. -Protein of interest coupled with GFP -observe fluorescence -One area is bleached with a laser, loses green fluorescence -remaining molecules move into photo-bleached area -the time to fluorescent recovery is measured
What is dark field microscopy?
-Light only directed at specimen from the side so only scattered light enters the microscope lenses -Image is bright object on dark background -Good for observing motility
What detergents are utilized to form micelles?
-SDS detergent -strong or mild detergent -can form micelle or liposome depending on concentration ex: sodium K pump can be purified and have proteins separated from bilayer and can be reconstituted in vitro -work on ratio of detergent to reconstitute pump to study function
What is cortical cytoskeleton?
-a common way in which a cell restricts the lateral mobility of specific membrane proteins is to tether them to macromolecular assemblies on either side of the membrane -cells have cortical cytoskeletal network beneath PM that imparts mechanical strength to cell -restricts diffusion of not only the proteins that are directly anchored to it -cytoskeletal laments are often closely apposed to the cytosolic membrane surface, they can form mechanical barriers that obstruct the free diffusion of membrane proteins -barriers called corrals
Bacteria can do what to adjust to temperature changes?
-adjust chains to accommodate fluidity -if temperature falls, synthesize more tails with cis double bonds
Describe the lipids in the lipid bilayer?
-amphiphilic -polar, hydrophilic heads -hydrophobic hydrocarbon tails -most abundant are phospholipids -1 chain saturated (no double bond, straight) -1 chain unsaturated (cis double bond, leads to kink in tail)
Membrane proteins are hard to work with. Why?
-amphiphilic nature -can be solubilized by detergents - when mixed with membranes, the hydrophobic ends of detergents bind to the hydrophobic regions of the membrane proteins, where they displace lipid molecules with a collar of detergent molecules -more soluble in H2O than lipids -detergent can have charged end or uncharged -concentrate of detergent in H2O low
What are different ways membrane proteins bind to the membrane?
-anchored to membrane - embedded in it -span membrane -attached by oligosaccharide linker -attached by noncovalent attachment to freely diffuse in membrane
What is a common problem with immunogold electron microscope? How do you circumvent?
-antibodies and gold particles may not penetrate resin and can only be detected at surface -lower-sensitivity and can't detect antigen deep in tissue only at surface fix: label specimen before embed it in plastic -additional silver or gold particles used and nucleated around very tiny gold particles
Describe the process of using fluorescent microscopy with antibodies?
-antibodies: detect certain antigen of molecule -can be labeled with fluorescent dye -amplifies fluorescent signal -either tag a primary or secondary antibody -if fluorochrome linked to secondary, stronger signal 1) direct: antibody linked directly to primary antibody 2). indirect immunocytochemistry: labeled to secondary -very sensitive, 2nd antibody detects unlabeled primary -can bind enzymes
What is limit of resolution have to do with aperture?
-aperture: measure of width of entry pupil on microscope -wider microscope opens its eye (more sharper image) - higher aperture: greater resolution
What is a disadvantage of light microscope?
-can enlarge image as much as you want, but won't always get best resolution -it is never possible to resolve two objects in the light microscope that are separated by less than about 0.2 um; they will appear as a single object -diffraction of waves can cause image to blur
The bilayer can function as complexes?
-can form a photosynthetic reaction center captures light energy and uses to pump protons across membrane -different functionalities -large systems work across domain of cell
Fluorescent microscopy can use multiple dyes to visualize multiple _____________?
-cell structures -ex: fluorescein: emits green light to view spindles in mitosis -rhodamine: emits red light -can use both to visualize cells
How do transmembrane proteins pass the membrane?
-chain crosses membrane in alpha helical confirmation - membrane spanning segment has nonpolar side chains arranged specifically and peptide bonds form H bonds with each other
How was diffusion in membrane discovered?
-created hybrid cell -fused mouse and human to create heterokaryon and use 2 antibodies (1 specific to mouse, 1 specific to human) -added, look at pattern of signal -at time 0, mouse on 1 side and human on other -at time 40 mins, mouse and human antibodies intermingled -indicates lateral diffusion of proteins
What shape can an individual phospholipid take on?
-cylinder: tends to form bilayer (to try to form least energetically expensive conformation) -cone shape: tends to form micelle
Some membrane proteins are only made in _____________?
-cytosol, others outside of cell
How is fludity of the membrane regulated?
-depends on temperature and composition -if made from single type of lipid, lipid changes state from liquid to gel depending on temp. -phase transition: change of state of bilayer to liquid to crystalline state at freezing temp. -occurs if tails are short or have double bonds -double bond makes kinks and decreases packing of lipids -unsaturated: liquid -saturated: solid
The lipid bilayer is asymmetrical due to its ?
-different charged heads -different charge needed for transport and signaling ex: RBC, made up mostly of phosphatidylcholine inner layer contains phosphotidylethanol
Animal cells exploit the asymmetry of the membrane to ___________?
-distinguish between live and dead cells -when cells undergo apoptosis, phosphatidylserine is translocated from inner layer to outer layer -signals for neighboring cells like macrophage to phagocytose dead cell and kill it translocation occurs 2 ways: 1) translocator that is thought to transport lipid from noncytosolic monolayer to cytosolic monolayer is inactivated 2)scramblase that transfers phospholipids in both directions of monolayers is inactivated
The bilayer can form ______________?
-domains -can form lipid rafts -cavoelae in PM enriched in spingolipids and cholesterol form rafts -can stabilize proteins
Cells confine proteins/lipids to ____________?
-domains -certain plasma membrane enzymes confined to apical surface -basal surface -lateral surface -different function wherever localized
Describe how membrane proteins diffuse in plane of the membrane?
-don't flipflop across membrane -capable of rotating around axis perpendicular to membrane -rotational diffusion: circular motion -laterally move as well (lateral diffusion)
What is a drawback of methods to discover membrane protein movement?
-drawback: only monitor large complexes of molecules in large areas of membrane not individual protein molecules -need: single particle tracking technique to track individual molecule by labeling with antibodies coupled to dyes
What is a transmission electron microscope?
-electrons are scattered by collisions with air molecules, air must first be pumped out of the column to create a vacuum -electrons are then accelerated from the filament by a nearby anode and allowed to pass through a tiny hole to form an electron beam that travels down the column. -Magnetic coils placed at intervals along the column focus the electron beam, just as glass lenses focus the light in a light microscope. The specimen is put into the vacuum, through an airlock, into the path of the electron beam - the specimen is usually stained-in this case, with electron-dense material, as we see in the next section Some of the electrons passing through the specimen are scattered by structures stained with the electron-dense material; the remainder are focused to form an image -can be viewed on phosphorescent screen -resolution: 100, 000 X better
What is the purpose of the plasma membrane?
-encloses its boundaries -to define boundaries and separate the inside of the cell from extracellular conditions -membrane doesn't allow mixing of contents with each other -also establishes a gradient for movement of compounds
What is Atomic force microscopy?
-extremely small pointed tip of silicon used, attached to springy cantilever arm to enable tip to move - measure mechanical force that tip touches -sense of tip conveyed to tip -probe scanned over surface, revealing imaging pattern -also can move molecules to unfold it to measure protein domain folding
What happens when detergent monomers are mixed with membrane?
-form micelles -hydrophilic AA chains embedded inside or hydrophobic -causes destruction of bilayer to bring into solution
How is the lipid bilayer fluid?
-free diffusion in bilayer -lipids have lateral diffusion, flip-flop or flexion -liposomes: transfection of mammalian cells, diffuse molecules through bilayer
What are found on the surface of the plasma membrane?
-glycolipids -contain sugar and exclusively found in noncytosolic layer -made of sphingosine -asymmetric distribution of glycolipids due to addition of sugar molecule in lumen of Golgi apparatus -sugars exposed on outside of membrane and have distinct roles ex: gangliosides have net neg. charge -in brain, needed for PM integrity and can protect membrane from harsh conditions -can change electric field to negotiate ion traffic across membrane -interact with lectins and proteins
Membrane proteins are _______________?
-glycosylated: oligo chains outside membrane decorates membrane extensively -sugar residue added in ER or Golgi -protein on one side of membrane also form disulfide bonds between cysteine residues -chains on non cytosolic side of cell -cytosolic region is reducing environment that breaks disulfide bonds
How do cells store lips?
-in lipid droplets that form rapidly in presence of fatty acids -cells can tap into adipocytes to use lipids as food source or membrane assembly -neutral (exclusively hydrophobic) lipids deposited between 2 layers of ER and aggregate into droplets, pinch off bilayer and bud off surrounded only by a single layer of lipids -not amphiphilic
How do you fix cells?
-in-tact tissues normally require fixation/sectioning due to their size -1). immobilize cell, kill it and preserve detail -fixatives can form covalent bonds between free amino groups of proteins and cross link and stabilize them 2) resins/waxes: tissues to soft so need medium, resins and waxes permeate tissue, cool and harden into block, sectioned with microtome into slivers -ribbon of section placed on glass slide, slip put on and observed
What is multiple image microscopy?
-increase resolution -take image of identical molecule and combine to produce average image to reveal structural details hid by noise -1000s of images -need to align images with one another -also called single-particle reconstruction
What enhances permeability-barrier properties of the membrane?
-inserts into the bilayer with its hydroxyl group close to the polar head groups of the phospholipids, so that its rigid, plate like steroid rings interact with-and partly immobilize-those regions of the hydrocarbon chains closest to the polar head groups
How is a light microscope arranged?
-lens have light shine through to direct image straight onto eye
What are 2 preparations of synthetic lipid bilayers?
-liposomes: spherical vesicles -black membrane: planar bilayers -measures permeability
What is a proteoglycan?
-long polysaccharide chain covalently linked to protein core -found mostly outside cell or spanning membrane
How can you use FRAP to discover lateral movement in bilayer? What about FLIP?
-mark protein with fluorescent molecule -label antibody in membrane -fluorescence observed then bleach area and wait for proteins to invade area and see if green fluorescence is reconstituted -indicates movement of proteins -FLIP: laser beam irradiates are to bleach all fluorescent molecules that move into it and gradually depletes surrounding membrane of fluorescent molecules
What spans the lipid bilayer?
-membrane proteins -transmembrane proteins pass through membrane and are amphiphilic in nature -hydrophobic regions interact with hydrophobic tails of lipids and hydrophilic regions exposed to water on either side of membrane
What is needed to view cells?
-need high level of magnification -cells hard to see by human eye
What is sphingomyelin?
-not built from glycerol, built from sphingosine backbone -long acyl chain with amino group and with 2 hydroxyl groups on one end -fatty acid tail attached to free amino group and phosphocholine attached to terminal OH group -free OH group contributes to polar properties of adjacent head group to form H bonds with other head groups lipids/molecules
What is photoactivation?
-observe high detail in cell -molecules move constantly -introduce molecule to monitor -synthesize inactive form precursor of fluorescent molecules, introduce it into cell and activate it suddenly at chosen site in cell by focusing light on it -inactive photosensitive precursors called cage molecules made for fluorescent molecules - a microscope can be used to shine strong pulse light from laser of specific wavelength at tiny region of cell and to control when and where molecule is photoactivated -light induced activation of inert molecule and causes it to activate
What are different kinds of lipids?
-phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine all have basic base structure and phosphate bound to different
How does the lipid bilayer seal the inner compartments of cell?
-planar bilayer edge exposed to water and forms configuration to hide tails The prohibition of free edges has a profound consequence: the only way for a bilayer to avoid having edges is by closing in on itself and forming a sealed compartment
What can be used in place of fluorescent dyes?
-radioisotopes -very specific (ex; 32P) -can be used to follow any process in cell -pulse-chase: DNA labeled, isotope decays and emits electrons -emits electrons which get captured by autoradiography and imaged
How do you assert image is similar to actual configuration in cells?
-rapid-freezing: utilizes aqueous system, cooled quickly, water components don't have time to arrange into ice so they go through glassy phase called vitreous ice -slam specimen on copper block and cool by liquid helium or plunging it into coolant -block either viewed directly by microscope or fractured -best way to view thin sections rapid-freeze the tissue, then replace the water, maintained in the vitreous (glassy) state, by organic solvents, and finally embed the tissue in plastic resin, cut sections, and stain( preserves the tissue in a condition very close to its original living state) -contrast depends on atomic number of atoms -higher atomic number, greater electrons scattered, and greater contrast -use salts or heavy metals to increase resolution
Describe the nature of the bilayer?
-serve as impermeable barrier to hydrophilic molecule -mostly hydrophobic -transmembrane proteins can be hydrophilic or hydrophobic and allow molecules to pass thru -fluidic nature
What is fluorescence microscopy?
-similar to a light microscope but the illuminating light is passed through one set of filters before the specimen, to select those wavelengths that excite the dye, and through another set of filters before it reaches the eye, to select only those wavelengths emitted when the dye fluoresces -glows against dark background -can detect proteins and be coupled with antibodies to serve as specific staining agents that bind to particular macromolecules to detect high degree of detail in cell
How do you increase contrast of image?
-stain cells -unstained area won't be see very clearly
What is cholesterol?
-sterol -rigid ring structure, to which is attached a single polar hydroxyl group and a short nonpolar hydrocarbon chain -orient themselves in the bilayer with their hydroxyl group close to the polar head groups of adjacent phospholipid molecule
What is bright field microscopy?
-the image is obtained by simple transmission of light through the object being viewed -light passing through object forms image directly
What is image deconvolution?
-the wavelike nature of light means that the microscope lens system produces a small blurred disc as the image of a point light source with increased blurring if the point source lies above or below the focal plane. -this is called the point spread function -first obtain a series of (blurred) images, usually with a cooled CCD camera, focusing the microscope in turn on a series of focal planes-in effect, a (blurred) three-dimensional image. -The stack of digital images is then processed by computer to remove as much of the blur as possible -optical sections imaged at diff. depths, stored, and reconstructed
How do you image cells using cameras?
-to increase our ability to observe cells in Iow light conditions, attach cameras with a charge-coupled device (CCD) - CCD cameras are often cooled to reduce image noise. -It is then possible to observe cells for long periods at very low light levels, thereby avoiding the damaging effects of prolonged bright light (and heat).
What is confocal microscopy?
-type of light microscope that produces a clear image of a given plane within a solid object -It uses a laser beam shines through confocal pinhole and serves as a pinpoint source of illumination -fluorescence emitted from the illuminated material is collected and brought to an image at a suitable light detector -pinhole aperture placed in front of detector in position confocal with illuminating pinhole precisely where rays emitted from illuminated point in the specimen come to a focus and scans across the plane to produce a 2D/ 3D optical section -emitted fluorescent light from in-focus point is focused at pinhole and reaches detector -emitted light from out-of-focus point is out of focus at pinhole and is largely excluded from detector -can be used to resolve 3D networks of fibers
How do you prevent nonspecific binding of polyclonal antibodies?
-use MaBs -have challenges: high level of specificity, if can't access epitope not useful
The cell has a coat?
-used to describe the carbohydrate- rich zone on the cell surface -this carbohydrate layer can be visualized by various stains, such as ruthenium red, as well as by its affinity for carbohydrate-binding proteins called lectins, which can be labeled with a fluorescent dye or some other visible marker
What is a hydropathy plot?
-used to predict the number and position of transmembrane segments in a given protein sequence -determines segments of proteins highly hydrophobic -very hydrophobic proteins span bilayer -plot mostly identifies segments containing about 20-30 amino acids with a high degree of hydrophobicity are long enough to span a lipid bilayer - not beta barrels as they are only comprised of 10 AA, and too small
What is phosphatidylinositol?
-various lipid kinases can add phosphate groups at distinct positions in inositol ring -creates binding points for to recruit proteins from cytosolic to membrane -embedded closer to inner layer of membrane to get activated by outer signals
How do imaging cells help not kill them?
-want to reduce light intensity so it doesn't kill cells -can use DIC imaging to enhance contrast
How is a single pass transmembrane protein made?
-while still in ER, transmembrane section of protein cleaved and GPI added to bind noncytosolic layer of membrane -transport vesicles transport it to plasma membrane -can release proteins by exposing it to phosphatidylinositol specific phospholipid C
What are 2 problems with the human eye?
1) can't see clearly in dim light 2) can't distinguish changes in light intensity against a bright background
What are 2 structures lipids can form in water?
1) micelle 2) bilayer
What are 2 kinds of membrane proteins?
1) peripheral: attached to membrane noncovalently, don't go into hydrophobic interior of membrane -can be released by exposing to ionic solvents or extreme pHs 2) integral: inserted into hydrophobic core of layer -can't be removed as easily
Most cells are 70% water and colorless? How do you observe the invisible details?
1). dye cells -depend on affinity -ex: hematoxylin: likes neg charged molecules like DNA 2)in situ hybridization -observe pattern of gene expression -requires probing 3).fluorescent probes: require absorption of light of specific wavelength at emits at longer wavelength light
What is a disadvantage of fluorescence microscopy?
1). only certain wavelength excites cell 2). can fade quickly when continuously illuminated -nowadays more stable fluorochromes -nanoparticles/quantum dots: can be excited to fluoresce through broad spectrum of blue light, keep shining for weeks
What is Negative staining?
A contrast-enhancing technique for the electron microscope in which a heavy-metal salt is used to create a reverse, or negative, image of the object. -finer details -molecule supported on thin film of carbon, mixed with heavy metal salt, thin film of metal salt covers carbon everywhere but -read 9.52
What is image processing?
Computer treatment of images gained from microscopy that reveals information not immediately visible to the eye
What is CMC?
Critical Micellar Concentration -this is when detergents form micelles in raised detergent levels -Detergent molecules rapidly diffuse in and out of micelles, keeping the concentration of monomer in the solution constant, no matter how many micelles are present.
What is green fluorescent protein (GFP)?
Fluorescent protein (from a jellyfish) that is widely used as a marker for monitoring the movements of proteins in living cells -newly made protein not fluorescent, but after an hour it undergoes post-translational modification and fluoresces -used as tag and fusion protein -can be reporter molecule coupled to promoter to detect activity of gene expression
The amphiphilic nature of lipids allows them to form bilayer spontaneously in ________________?
H20 -heads outside, tails tucked away in membrane -hydrophilic polar end soluble in water, hydrophobic tails non-soluble as they are uncharged and can't form energetically favorable connections with H20 -hydrophobic tails force water to forms cage that surrounds hydrophobic molecule
What is a microelectrode?
Piece of fine glass tubing, pulled to an even finer tip, that is used to inject electric current into cells
What is the distinction between detection and resolution?
Resolution is defined as the smallest distance by which two objects can be separated and still be distinguished. Detection is the ability to determine the presence of an object
What are common fixatives in EM?
Since the specimen is exposed to a very high vacuum in the electron microscope, living tissue is usually killed and preserved by fixation-first with glutaraldehyde, which covalently cross-links protein molecules to their neighbors and then with osmium tetroxide, which binds to and stabilizes lipid bilayers as well as proteins -permeated in block and cut into slivers to view on slide
What is fluorescence resonance energy transfer (FRET)
Technique for monitoring the closeness of two fluorescently labeled molecules -2 molecules labeled with fluorochrome -emission of 1 fluorochrome overlaps with absorption spectra of other -protein far away: no emission -close together: excites fluorescent light
What is the limit of resolution?
The minimal separation between two objects at which they appear distinct -relies on wavelength of light and numerical aperture of lens used
What is optical microscopy?
The most important method of fiber identification that involves polarizing light and comparison microscopes -less damaging to cells as does not require disruption of cells
What is Scanning Electron microscopy?
Type of electron microscope that produces an image of the surface of an object -directly produces 3D image of surface -smaller/cheaper than TEM -uses electrons scattered or emitted from surface of specimen -specimen fixed, dried and coated with thin layer of heavy metal (or frozen) -specimen scanned with narrow beam of electrons -quanity of scattering electrons used to control intensity of second beam, which moves in synchrony with primary beam to form image on TV screen -detector captures scattered electrons for high depth of detail -angles give shadows -can observe nuclear pores
What is Electron microscope (EM)?
Type of microscope that uses a beam of electrons to create an image. -better for smaller structures -requires stringent level of preparation of specimens -rapid freezing of cells to preserve cell features and digital analysis to reconstruct cell
When are light waves in or out of phase?
in phase: 2 trains of waves reach same point and are said to be in phase -crest of both waves in line, troughs in line -reinforce each other and make image brighter -out of phase: crest of one wave in line with trough of other -interfere with one another and cancel out or obscure part of image
What is Light-emitting?
ion sensitive indicators whose light emission reflects local concentration of ion -can detect concentration of ions in cells
The lipid bilayer is made up of _____________ and _____________?
lipids and proteins -thin film of lipid (fatty) molecules and proteins joined by noncovalent bonds -animal cells made up of 50% lipids (billion lipids in a membrane) -arranged in bilayer 5nm thick
Besides lipids, bilayers are also made up of ___________?
proteins -myelin membrane: 20% protein in layer -chloroplast membrane: 75% typically half protein, half lipid
Cameras are used in imaging to?
reduce noise and observe cells
What are single pass and multipass transmembrane proteins?
single pass: pass membrane once multipass: pass multiple times (either Alpha helices or Beta barrels)
Microscopes are used to ?
study and visualize cells
Multipass proteins are newly ________________?
synthesized -fold slowly and contacts between helices displace lipid molecules away from helice
What is the best resolution image in a light microscope?
violet light and numerical aperture of 1.4 microscope can achieve limit of resolution of 0.2 um
What sets the limits of visible light?
wavelength of light
Lipids are randomly distributed and form ____________________ forces to pull hydrocarbon chains together?
weak van der Waals forces between molecules -however not strong
Transmembrane alpha helices interact with ______________?
with one another to form homodimerize and held together by interactions -held together by strong and specific interactions between to a helices -sequence of hydrophobic AAs determines protein interactions
