Quiz 1 Flashcards
(123 cards)
1
Q
What are the major life domains?
A
- Bacteria
- Archaea
- Eukarya
2
Q
What are good bases for comparing distantly related species? What about closely related species?
A
- Ribosomal DNA
- DNA sequencing
*Tree is based on ribosomal DNA
3
Q
Major differences between eukaryotes and prokaryotes
A
- Prokaryotes lack membrane bound organelles. Eukaryotes have many single and double membrane bound organelles
- Prokaryotes are typically smaller than eukaryotes
- Eukaryotes divide by mitosis or meiosis and prokaryotes divide by binary fission
4
Q
Similarities in prokaryotes and eukaryotes
A
- Plasma membrane of similar composition and structure
- Genetic information encoded by DNA using the same genetic code
- Similar mechanisms for transcription and translation
- Same basic metabolic pathways (e.g. glycolysis and TCA)
- Similar apparatus for conserving chemical energy as ATP
- Similar mechanisms for photosynthesis (cyanobacteria vs chloroplast)
- Similar mechanisms for synthesizing and inserting membrane proteins
5
Q
Features found in eukaryotic but not prokaryotic cells
A
- Cells have nucleus and cytoplasm, separated by nuclear membrane
- Complex chromosomes composed of DNA and proteins capable of compacting DNA into mitotic structures
- Complex membranous organelles (ER, Golgi, lysosome)
- Specialized structures for respiration (mitochondria) and photosynthesis (chloroplast)
- Complex cytoskeleton (microfilaments, microtubules, intermediate filaments)
- Complex cilia and flagella
- Endocytosis
- Cell division
- Sex reproduction
- Presence of two copies of genes (diploid), one from each parent
6
Q
Resolution (D)
A
Defined as the smallest distance between two points at which the points can be recognized as two entities instead of one
7
Q
The smaller the value of D, the higher or lower is the resolution?
A
The smaller the D, the greater teh resolution
8
Q
What would make a high-resolution objective?
A
To make high resolution microscopy, lenses are designed to work w/ oil between the lens and the specimen since oil has a higher refractive index (N)
To maximize the angle alpha, and hence sinalpha, the lenses are also designed to focus very close to the tin coverslip covering the specimen
9
Q
Numerical aperature (NA)
A
N sina
10
Q
N
A
The refractive index of the medium between the specimen and the objective lens (usually air)
11
Q
Diffraction limited
A
The limit of resolution of a light microscope is 0.2 micrometers or 200
12
Q
Relative Resolutions of different types of microscopy
A
- Light microscopy: can see down to 200 nm or at the organelle level
- SEM and Fluorescence microscopy can see down to the protein level
- TEM can see down to the electron level (1 nm)
13
Q
How is the resolution limit related to the type of applications for each microscopy?
A
The resolution limit is directly proportional to wavelength of light. The shorter the wavelength is, the greater resolving power (D) you have. Electron beams have really short wavelengths which is why they are able to see things at a greater resolution
14
Q
Confocal microscopy
A
Uses optical methods to obtain fluorescence images from a specific focal plane and exclude light from other planes
15
Q
Point scanning confocal microscopy
A
Light path of a single-wavelength point of light from an appropriate laser reflects off a dichroic mirror and balances off two scanning mirrors and passes through
Laser beam –> mirror –> specimen –> fluorescence from specimen bounces back –> mirror –> pinhole
16
Q
Spinning disk confocal microscope
A
Light path from the laser is spread to illuminate pinholes on the coupled spinning disk, the first consisting of microlenses to focus the light on pinholes in the second disk. The excitation light passes through the holes in the spinning disk and is then bounced of the mirror into a sensitive digital camera. The pinholes in the disk are arranged so that as it spins, it rapidly illuminates all parts of the specimen several times
17
Q
Benefits of the two types of confocal microscopy
A
- Spinning disk: allows you to take multiple pictures of the specimen at one time, so it is faster and ideal for live photos
- Point scanning disk: allows you to take one picture of one area of the specimen at a time, allowing for you to take images of thicker samples. Slower process
18
Q
What is the role of the “dichroic mirror” in fluorescence microscopy
A
It allows the mirror to separate excitation and emission lights, clearly enabling detection of fluorescence w/o interference from the excitation light source. If not, the image would appear blurry
19
Q
Recognize the different types of light microscopy
A
- Brightfield is 2D with no contrast
- Phase Contrast is 2D with contrast
- Fluorescence is 3D
20
Q
Fluorescence microscopy
A
A chemical is said to be fluorescent if it absorbs light at one wavelength (excitation) and emits at a specific longer wavelength
*Two or even 3 fluorescent dyes of different colors can be visualized
21
Q
Wide field/conventional fluorescence microscopy vs confocal microscopy
A
- A conventional fluorescent microscope leads to blurry images because of the background fluorescence from above and below the plane
- Confocal microscopes only detect fluorescence from molecules in the focal plane, generating very thin optical sections
– Optical sections from confocal can reconstruct a 3D object
– pinhole blocks out any out of focus light that isn’t a part of the plane
22
Q
Both “conventional microscopy” and “fluorescence microscopy” are forms of light microscopy. Why is fluorescence being useful for 10X smaller dimensions, down to about 10 nm?
A
it harnesses the specific emission of light from fluorescently labeled molecules within a sample, enabling the visualization of very small structures or specific targets with high sensitivity, essentially “highlighting” them against a dark background, whereas conventional microscopy relies solely on light absorption and scattering from the sample, limiting its resolution to larger structures.
*Not related to wavelength
23
Q
Deconvoluted microscopy
A
Deconvolution microscopy uses calculated point-spread functions of out-of-focus light to
computationally remove fluorescence contributed by out-of-focus parts of the sample. AFTER the images have been aquired
24
Q
Total internal reflection fluorescence (TIRF) microscopy
A
For imaging molecules within a restricted focal plane near the coverslip
*imaging things on the surface
25
Two-photon excitation microscopy
- For imaging deep tissue samples such as the brain of an alive mouse
*use energy of two photons instead of one of lower energy wavelengths because lower energy penetrates tissue deeper
26
Light sheet microscopy
- For rapidly imaging a large volume, for example in transparent living tissues
27
Fluorescence recovery after photobleaching (FRAP)
- To reveal cellular dynamics
28
Forster resonance energy transfer (FRET)
- For studying protein-protein interaction
29
Super resolution microscopy: localization microscopy
If you could image each fluorescent molecule individually and find the center of each spot, you could "beat" the resolution limit and separate spots that are too close for conventional microscopy
*For every image, a small number of GFP molecules are activated. Each GFP will emit thousands of photons that can be collected as a Gaussian curve centered on the location of the emitting GFP. Reiterations hundreds of times to excite other GFP molecules generates a high resolution image
30
Super-resolution microscopy: Structured illumination microscopy (SIM)
The sample is illuminated by a pattern of light and dark stripes and several images are taken as the illumination is rotated, generating interference patterns that can be mathematically rec
31
Super-resolution microscopy: Stimulated emission depletion microscopy (STED)
The sample is scanned as in point-scanning microscopy with a very small point of light generated by an emission laser and confined by a donut shaped stimulated emission depletion zone
We do STED microscopy to achieve super-resolution imaging by using a second laser to deplete fluorescence around the focal point, leaving only a small region emitting light, which sharpens the resolution beyond the diffraction limit. So by having the depletion zone, you remove excess fluorescence and focus in on one point at a time which gives you higher resolution
32
Two ways you can visualize specific proteins w/ fluorescence microscopy
- Express a fusion protein that contains a fluorescent protein
- Detect the protein of interest using antibodies: immunostaining
33
GFP
- A protein can be fluorescently tagged genetically with GFP
- Eleven strands form a cylindrical Beta-barrel to protect the internal fluorochrome
34
Key features of GFP
- Direct detection: no additional cofactors or proteins needed
- Species independent: works in almost everything
- Long half life
- Folds independently
- Can be chemically fixed and still fluoresces
- Has no localization signals, by itself, it resides in the cytoplasm
35
How to incorporate GFP into the genome
To make GFP tagged gene, the gene of interest and GFP have to be in the same reading frame. Usually, the function of the protein is fine even when it has GFP on the front or back of it
36
Enhanced GFP
Brighter, better at folding, codon optimized for human cells
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Superfolder GFP
Rapid folding, more stable, more reliable tag
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Color variants
*don't have to say too much about this, just that GFP has different colors now that have different excitations
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Monomeric GFP
GFP can dimerize, meaning that if it met another GFP that was bound to a protein, they could get stuck together. This would ruin the trafficking. Therefore, monomeric GFP was created because it has the ability to dimerize. Has the prefix m.
40
Photoactivatable GFP or PA-GFP
When PA-GFP proteins are photoactivated, you can directly measure the mobility of the labeled proteins. This allows you to only activate a small number of proteins at one time so you can make observations and see patterns more clearly
41
Different sensors
- pH sensor
- Redox sensors
- Metal ion sensors
*Don't need to know too much information about this, just need to acknowledge that sometimes the GFP molecule must be fused to different things to allow for detection
42
Td tomato
- Sometimes you want to create a dimer, this is where you would use something like Tdtomato. This means you have two copies of the tomato protein to force them to dimerize and track protein. This is advantageous because it allows for it to be brighter.
43
Photoactivatable proteins
Irreversible.
Can use photoactivation light to turn on the protein's fluorescence
44
Photoconvertible proteins
Reversible
Can switch protein fluorescence on or off
45
Photoswitchable proteins
Switch colors of the protein (like from green to red)
46
What does Td stand for
Tandem dimer
47
Compared to standard GFP fused proteins, what kind of novel information could one derive by observing a photoactivatable GFP attached to a protein in living cells?
Allows you to see dynamics much better so you can look at trafficking
48
Structure of an Antibody
IgG structure: two fold symmetric structures composed of two identical heavy chains and two identical light chains
- Chains attached by disulfide bonds and glycoprotein (for the heavy chain)
49
Fragmentation w/ proteases
Yield fragments that retain antigen binding capacity
- You can use Papain and end up with two, monovalent Fab portions
- You can use Pepsin and end up with one, bivalent Fab
50
Fab
Stands for antigen binding fragment which can bind a single antigen molecule. Therefore, it is a monovalent fragment
51
Fab2
Exhibit bivalent binding
52
Fc
Fc is incapable of antigen binding. It is called Fc because of its ease
of crystallization
53
Complementarity Determining Regions
Each heavy chain or light chain contains 3 hypervariable regions. These hypervariable regions are in close proximity in 3D and make contact with antigen.
54
Clonal selection of B lymphocytes
- Each individual B cell is programmed to make only a single molecular species of antibody. If the cell encounters an antigen and binds. it is stimulated to proliferate clonal expansion
b cell matures in that the antibody changes from being on the surface to being secreted
Complex antigens can present several molecularly distinct features that can stimulate clonal expansion of diff B cells - yields a polyclonal mixture of antibodies
55
Epitope
The part of the antigen molecule that binds to an antigen specific receptor on B or T cells or to antibody. Large protein antigens usually possess multiple epitopes that bind to antibodies of different specificity
*Can be linearized (denatured/recognized by its linear sequence of amino acids or primary structure)
Conformational epitope (folded/the sequence that comes into contact with CDR of an antibody)
56
Polyclonal
Many different clones of B cells were stimulated to proliferate and secrete IgG molecules, each to a different epitope
57
Hybridoma cells
B cells in a clonal population die out in culture; however, B cells can be fused with myeloma cells. These hybrid cells can be expanded to large populations and grow forever. Such a clonal population of B cell hybridoma cells secretes monoclonal antibodies
57
Is it usually an advantage to use a monoclonal or polyclonal antibody?
It is often a big advantage to use a polyclonal antiserum which has Abs that recognize epitopes all over a protein, not just one MaB
58
Primary antibody
Recognizes the antigen
58
Secondary antibody
Recognizes the Fc portion of the primary antibody. Usually chemically linked to a fluorophore or enzyme
58
Generation of monoclonal antibodies through hybridoma
- Inject a mouse with the antigen to induce B cell production
- Immortalize B cells by fusing them with myeloma cells to make hybridma cells
- Screen for the desired clone using antigen
59
Two types of electron microscopy
- TEM
- SEM
60
Scanning electron microscopy (SEM)
Electrons scattered off the specimen metal coating and are collected by the detector (similar to an eyeball that can see from all angles)
60
Transmission electron microscope (TEM)
Transmitted electron beam pass through specimen and hits the detector. SAMPLES MUST BE THIN so that all your electrons are not absorbed
61
How is EM different from light microscopy?
EM must be done in a vacuum since air molecules can interact with electrons
62
How is the contrast generated in EM?
In electron microscopy, contrast is generated by differences in the scattering of electrons as they pass through or are reflected by the sample, with denser areas scattering more electrons and appearing darker. Remember, the sample is coated with heavy metals which is why the electrons scatter once they pass them or are reflected off.
63
Negative stain TEM (for small particles)
Negative stain EM is just for small particles. The stain pools around the structure, hence called negative. Stain shows OUTLINE of particle, often plus some ultrastructure where stain partially penetrates structure, Typically, protein arrays show ultrastructure but membranes do not
ultrastructure refers to the fine details of the internal organization or components of the particle, visible at the molecular or subcellular level, typically revealed when the stain partially penetrates the structure.
63
What are some advantages and disadvantages of negative stain EM?
Advantages of negative stain TEM:
- Provides high contrast for small particles.
- Preserves the overall shape and structure of the sample.
Disadvantages:
- Limited to small particles.
- Stain may only reveal surface details, not internal structures.
(membranes)
63
Make sure you can tell apart TEM and SEM microscopy
- TEM is used for thin sections only, and has a 2D look to it
- SEM has a 3D look to it
64
Cryo-EM
*Powerful technique for studying structures of large protein complexes like the nuclear pore
- An aqueous suspension of a sample is applied to a grid of extremely thin film, frozen in liquid nitrogen. By computer-based averaging of hundreds of images, a 3D model can be generated almost to atomic resolution
- The specimen holder is tilted in small increments around the axis perpendicular to the electron beam; thus, images of the object viewed from different directions were obtained, and computationally into a 3D recnstruction
65
Immuno-EM
*make sure you can recognize this type of microscopy, you would see obviously black dots that are gold particles
- Was developed to use antibodies to localize proteins in thin section sat the electron microscope level. To make the antibody visible, it must be attached to a electron dense marker, like gold particles coated with protein A. Protein A binds the Fc segment of all antibody molecules.
66
FACs
A fluorescence activated cell sorter separates cells having different levels of fluorescence based on how they were labeled
*be able to interpret a flow cytometry plot
67
Differential centrifugation
Separate contents by rate of sedimentation. A fairly low speed pellets the nucleus, the largest organelle. The next highest speed sediments the mitochondria, chloroplasts, lysosomes, and peroxisomes. Subsequent centrifugation results in the deposition of plasma membrane, ER, and ribosome. The only thing that is left in the supernatant is the cytosol
68
Methods to break cells to release its contents
- Detergent
- Shearing (physical force such as sonication)
69
In differential centrifugation, what does sedimentation property depend on?
Size, shape and density (but really size)
70
Detergent vs Shearing
- With detergent, most protein-protein interactions remain intact, but membranes are dissolved and therefore most organelles are ruptured
- With shearing, harsh methods disrupt organelles. Mild methods usually require cells to be osmotically swollen first by exposure to hypotonic conditions
71
Equilibrium Centrifugation
An impure organelle fraction obtained by differential centrifugation can be further purified by equilibrium centrifugation, which separates cellular components according to their density.
*For instance, they would be in a solution of sucrose that has increasing density towards the bottom
* Size is irrelevant, spinning longer or harder once equilibrium has been reached is pointless since it will not change the profile of the bands
72
SDS Page
Separates proteins, primarily separates on the basis of their masses
1. Denature proteins with SDS, a negatively charged detergent that denatures proteins and coats each one with negative charges proportional to the mass of the protein.
2. Use gel electrophoresis, the electric field drives SDS protein complexes through the gel. Small complexes move faster than large complexes. Proteins separated into bands according to their size as they migrate
3. Stain separated protein bands with a dye for visualization
73
Western blotting
Can detect specific proteins and their binding partners
1. Transfer/blot proteins separated by SDS Page of electrophoresis onto a porous membrane that avidly binds all protein
2. Incubate membrane with a solution of an antibody specific for the protein of interest, which binds to a specific protein on the blot
3. Incubate membrane with a solution of a second antibody specifically that bin dsto the first Ab. This second Ab is covalently linked to a detectable label such as an enzyme or radioactive isotope
4. Detect label on second Ab, revealing location of protein bound by primary antibody
74
Do linear or conformational epitopes work better for western blotting?
Linear epitopes because conformational epitopes may be denatured by the SDS
75
Immune precipitation (IP) of a protein or protein complex
1. Break cells with a non-ionic detergent
2. Spin out debris in centrifuge
3. Add antibody to protein of interest and incubate 1 h in cold
4. Add Protein A (same protein A that binds to the Fc portion of antibodies) covalently couples to agarose beads and incubate for 1 h
5. Collect antibody-antigen complex by centrifugation. Wash extensively
6. Dissolve complex and denature proteins by boiling in SDS
7. Run SDS Page and detect protein bands
76
Mass Spectometry
Powerful technique for characterizing proteins, especially for determining the mass of a protein or fragments of a protein. This method permits the accurate direct determination of the ratio of the mass (m) of a charged molecule to its charge (z), or m/z.
77
Is mass spectrometry accurate?
Yes, it is very accurate
78
What do the peaks represent in mass spectrometry data?
The number at the peaks of the spectrometry data are the m/z ratio that determine the protein, these peaks can be broken down with inert gas to derive the AA sequence of the protein
79
Principles for primary mammalian cell culture
DNPCSMF
1. Require certain density: contact inhibition of growth
2. Require nutrients
-- inorganic salts, sugars, amino acids, vitamins, fatty acids
3. Require protein serum: serum dependence
-- such as serum (signaling)
4. Growth ability depends on cell type
5. Need to adhere to solid surface: anchorage dependence
6. Have a finite life span: mortality
7. Cells can transform to divide forever
80
Mortality of primary fibroblast
- Primary cells will divide a finite number of times, then cease growing and eventually die (around 30 days). Very rare cells in a population of primary cells may undergo spontaneous oncogenic mutation leading to oncogenic transformation. Such cells are said to be transformed.
These cells will grow indefinitely and cells that are immortal are called a cell line
81
Cell senescence
Cells cease dividing
82
Oncogenic transformation
Very rare cells in a population of
primary cells may undergo spontaneous oncogenic mutations, leading to oncogenic
transformation. Such cells, said to be oncogenically transformed, or simply transformed, are able to grow indefinitely (later lecture on cancer)
*not common in humans, cell lines from humans are typically derived from cancer
83
Cell line
Cells that are considered immortal
84
Transient Transfection vs Stable Transfection/Transformation
Transient and stable transfections differ in whether the recombinant vector DNA is or is not integrated into the host genome
85
Transient transfection
Protein is expressed from the plasmid
86
Transformation/ stable transfection
Protein is expressed from the plasmid that recombined into the genome
*After the electroporation process, these cells have to be selected for
87
Viral origin of replication
Enables replication of the plasmid or DNA sequence by providing a starting point for the viral or host DNA replication machinery
88
Phospholipids
Building blocks of biomembranes. All phospholipids are amphipathic
89
Leaflet
Each phospholipid layer in the structure is called a leaflet
90
Membrane topology in eukaryotic cells
A cellular membrane has a cytosolic face and a exoplasmic face. The exoplasmic face is directed away from the cytosol, towards the extracellular space or external environment, and it defines the outer limit of the cell. The cytosolic face of the plasma membrane faces the cytosol
- The exoplasmic face of an organelle contacts with the internal aqueous space, or lumen
91
What organelles have double membranes
Nucleus and mitochondria (chloroplasts in plants)
92
Inner membrane and outer membrane
This only refers to organelles that have double membranes. There is an inner and outer one
93
Biomembrane is composed of 3 classes of amphipathic lipids
Phosphoglycerides, sphingolipids, and sterols
94
Phosphoglycerides
- Glycerol backbone
- Two hydrophobic fatty acyl chains that vary in length and saturation
- Polar head groups: PC, PE, PS, PI
95
Sphingolipids
- Amine group that connects various fatty acyl tails
- Sphingomyelin: has a phosphocholine headgroup. Hydrophobic part is longer than typical glycerol phospholipids
- GlCer: has a glucose head group
96
Sterols
Head group: single polar hydroxyl
Tail: conjugated 2 ring hydrocarbons and short hydrocarbon chain
97
Which phosphoglyceride head groups have charges?
PS and PI
98
Fluid mosaic model
The phospholipid bilayer behaves in some respects like a 2D fluid, with individual lipid molecules able to move past one another as well as spin in place
99
Asymmetry on outer vs inner leaflet
Outer: all glycolipids, most SM, and OC
Inner: PS, PI, and PE
100
How do we compensate for the negative charge of PS and PI?
Transmembrane proteins will have positively charged residues on the side that faces the cytosol. (Lys, Arg)
101
How is cholesterol distributed along the leaflets?
Evenly
102
Phospholipases
Enzymes that cleave the ester bonds between the acyl chains or the head group and the lipid
103
How are the phospholipases named?
They are named after the bond they break. Each phospholipase has a unique ester bond to break
104
SM and cholesterol
SM associates into a more gel like and thickness bilayer than phosphoglycerides. Because SM tails are already optimally stabilized, the addition of cholesterol has no effect on the thickness of the bilayer
105
Phosphoglycerides and cholesterol
Cholesterol decreases membrane fluidity and also increases bilayer thickness
106
Membrane curvature
- Dictated by the size of the head group and the length of the tails
PC: large head group, long tails --> leads to a cylindrical shape and it is flat when they are lined up
PE: small head, long tails *--> leads to curvature when they are lined up
107
Phase transition
Bilayer can go from melting to freezing or in other words liquid to gel
108
Movements of lipids in bilayer
1. Lateral diffusion
2. Flexion
3. Rotation
4. Flip flop
Lateral diffusion moves lipids mean micrometers in seconds while flip flops rarely occur and it could take day
109
Does heat impact the phasing of the lipid bilayer?
Yes, heat transform it from a gel to liquid structure
110
Cholesterol as a buffer
At high temperatures, cholesterol stiffens the membrane by restraining the movement of phospholipids, preventing the membrane from becoming too fluid.
At low temperatures, cholesterol loosens the membrane by disrupting the tight packing of phospholipids, preventing the membrane from becoming too rigid.
111
Tail length on membrane fluidity
The longer the tail, the more stiff the membrane packs the phospholipids together
112
Saturation on membrane fluidity
Increase in unsaturated tails leads to more fluidity
113
Many fish are rich in polyunsaturated fatty acids (PUFAs). Why?
Because the cold temperatures would force their plasma membranes to be too rigid, so to compensate for this, they have many unsaturated tails to allow for fluidity
114
Integral membrane proteins
Integral membrane proteins, also called transmembrane proteins, span a phospholipid
bilayer and comprise three domains. The cytosolic and exoplasmic (or extracellular)
domains have hydrophilic exterior surfaces that interact with the aqueous environment
on the cytosolic and exoplasmic faces of the membrane. In contrast, the membrane-
spanning segments (or transmembrane domain) usually contain many hydrophobic
amino acids whose side chains protrude outward and interact with the hydrophobic
hydrocarbon core of the phospholipid bilayer
115
Lipid-anchored
Lipid-anchored membrane proteins are bound covalently to one or more lipid
molecules. The hydrophobic tail of the attached lipid is embedded in one leaflet of the
membrane and anchors the protein to the membrane. The polypeptide chain itself does
not enter the phospholipid bilayer.
116
Peripheral membrane
Peripheral membrane proteins are bound to the membrane either indirectly by
interactions with integral or lipid-anchored membrane proteins or directly by
interactions with lipid head groups. They are often operationally defined by being
stripped from membrane with high salt
117
