Final Flashcards
(391 cards)
1
Q
Explain how the connection of integrin to fibronectin is formed
A
Gradually, the initially formed fibronectin fibrils mature into highly stable matrix components by covalent cross-linking
2
Q
Explain the structure of the IgCAM
A
Consist of numerous transmembrane domains that contain multiple Ig domains in the extracellular regions
3
Q
What are JAMs present in?
A
Tight junctions
4
Q
What do ICAMs bind to? What cells are they on?
A
ICAMs on endothelial cells mediale heterophilic interactions with integrins
5
Q
What do selectins mediate the trafficking of?
A
They mediate the trafficking and regulation of white blood cells
6
Q
What type of interactions do selectins mediate? Cell to ___
A
They mediate transient cell to cell interactions in the bloodstream.
7
Q
What is the name of the integrin on the leukocyte?ICAM?
A
aLb2
ICAM 2
8
Q
What are the ways to get the leukocyte to express the integrin molecule?
A
- Binding of PAF to receptor
- Strong chemokines
9
Q
What are the two ways exogenous DNA can be introduced? Is this non-homologous or homologous recombination?
A
- In cell culture: it can be introduced through transfection
- In germ line: foreign DNA can be injected into the fertilized egg
This is non-homologous recombination in both instances
10
Q
How do you make a transgenic mouse (diagram)
A
- Fertilized egg is injected with foreign DNA before the two pronuclei fuse
- The egg is then transferred into a foster mother
- About 10-30% of the offspring will have foreign DNA incorporated
11
Q
Are transgenes randomly inserted? How are they controlled?
A
Yes, transgenes are randomly inserted in the genome via the nonhomologous pathway. But, they are usually engineered so that they contain a regulatory promoter so that you can control which tissue it is expressed in and when.
12
Q
What is the main goal of using yeast with homologous recombination?
A
To modify the endogenous genes to have your desired sequences
Applies if you want to do knock ins or knock outs
13
Q
Why are yeast so good to use for these experiments?
A
- They readily take up exogenous DNA in the right conditions
- They efficiently incorporate the DAN through homologous recombination
14
Q
For CRISPR, which repair pathway is considered for knock outs? Which repair pathway is considered for knock ins?
A
- NHEJ pathway is considered for knockout mutations
- HDR is considered for knock in mutations
15
Q
Can you explain how to create a CreLox mouse, including the parents?
A
- One parent is homozygous for the loxP sites - this mean that every cell has a loxP site
- The other parent is heterozygous for the Cre protein. The Cre protein is also controlled by being placed in front of a cell specific promoter. For instance, if the Cre is next to a Lung promoter, the Cre protein will only be transcribed in lung cells
- Offspring should have both Cre and Lox expressed. Cre will only activate if they are in a cell that has the cell specific promoter activated.
16
Q
How do you know that sequences are homologus?
A
They share enough similarities to suggests that they have a common ancestor.
17
Q
Which relationship between proteins in a family are the most similar in function?
A
Orthologous genes
18
Q
What is the diffracted limited for light microscopy?
A
200 nm or 0.2 microm
19
Q
What is the alpha in the resolution equation?
A
This is the angle at which the cone of light hits the specimen
20
Q
How can you tell you’re looking at phase contrast microscopy?
A
The specimen will have clear contrasts with its background
21
Q
Explain DIC microscopy, how can you tell that you’re looking at it?
A
DIC is analogous to phase contrast microscopy, with portions of the image being enhanced
by differences in refractive index, but the images have a “three dimensional” look
22
Q
Typically in fluorescence microscopy, light is absorbed at a higher wavelength and emitted at a longer, wavelength. What is the exception to this?
A
Two-photon microscopy
23
Q
Which is better, wide field microscopy or confocal microscopy? Why? Explain what confocal microscopy is
A
Confocal microscopy is better. Confocal microscopy is a type of fluorescent microscopy that focuses on only detecting fluorescent molecules in one focal plane. It can achieve this using a pinhole, this shows only one focal plane.
Wide field microscopy detects fluorescent proteins from both aboce and below the focal plane - causing a hazy photo
24
Q
What are the two types of confocal microscopy?
A
- Spinning disk microscopy: allows you to image cells live in dynamic movement
- Point scanning microscopy: good for deep samples?
25
Explain two photon microscopy:
Two photons of the lower-energy wavelength (e.g., 960
nm) arriving almost instantaneously are both absorbed and induce the electron
to jump to the excited state. Vibrational relaxation (black dashed arrow) back to
the ground state emits a photon (507 nm), as in conventional point-scanning
confocal microscopy
26
Light Sheet Microscopy - what is it good for?
Light sheet microscopy is good for visualizing large live samples. However, the samples need to be transparent
27
What are all the methods for fluorescent micrscopy?
- Deconvolution microscopy
- TIRF
- Two photon excitation microscopy
- Light sheet microscopy
- FRAP
- FRET
- Confocal microscopy
28
Define simple sequence repeats? Are they typically long or short?
Composed of perfect or near perfect duplications of short DNA sequences
29
Is simple sequence DNA in tandem repeats?
Yes, for microsatellites, for instance, they are usually in a repeat of 150 bp
30
Draw the graph for the mobile DNA elements in eukaryotes. In addition, explain the process of retrotransposons
Retrotransposons start by using RNA polymerase to make an RNA intermediate. The RNA intermediate is then converted back to a DNA intermediate via reverse transcriptase. The DNA intermediate is then inserted into the site
31
Draw out an IS element? Who was the first IS element discovered in?
It was discovered in E.coli. Although eukaryotes can have transposons, they more often have retrotransposons
32
Why do transposons lead to unequal crossing over? What's an example
Normally, crossing over should happen between homologous pairs and they're lined up based on having similar DNA sequences. DNA transposons have similar DNA sequences and may cause homologous pairs of DNA to line up to pair up the transposons, but the transposons aren't even in the same part of the DNA on both chromosomes. This leads to unequal crossing over
*Fibronectin
*B globulin
33
Can you explain what exon shuffling is? What about this is different from unequal crossing over?
Transposons can lead to the recombination/crossing over of DIFFERENT genes as opposed to genes that are the same and should pair homologous (this creates repeats). The transposons can insert themselves into random genes and allow for the recombination of the random genes due to the similar sequences of the transposons.
Essentially, recombination is happening with genes that shouldn't pair, leading to new genes.
34
Which types of transposons have the ability to carry nearby exons when they cut themselves out
DNA transposons and LINES
35
What is the charge of histone proteins?
Positively charged
36
What are the ways you can discriminate chromosomes from one another?
Based on size, shape and staining
37
What are the two ways of visualizing chromosomes to make a karyotype?
- Giemsa (dark and white banding)
- Chromosome painting
38
Explain how chromosome painting works?
Chromosome painting is a variation of FISH. The idea is that you have probes that are complementary to the chromosome and have fluorescent dyes attached to them. Different chromosomes are pre decided with what fraction of dye they are going to get, so when you see it, you know what chromosome they are. It is then put under a fluorescent microscope
39
Explain telomeres and fix your confusion about them
All cells will naturally start with elongated telomeres. This is a repeat of TTAGGG. The 3' end of the GGG strand will have 12-16 more nucleotides to bind proteins that prevent exonucleases from eating at the DNA. Telomeres loop
40
How does telomerase work?
Telomerase is a reverse transcriptase that elongates the TEMPLATE strand of the DNA by adding TTAGGG. This gives room for an RNA primer to be added to finish the Lagging strand synthesis
41
Explain next generation sequencing and Chip seq
- Amplification phase includes having a linker that will bind to the primers on the plate and amplify
- After amplification, one of the strands is cut and new primers are added so that ddNTPs with fluorescence can be added and you can tell which base it is.
ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) is used to find out where specific proteins (like transcription factors or histone modifications) bind on the DNA across the whole genome. (which genes are being transcribed the most)
Crosslink proteins to DNA (usually with formaldehyde) to "freeze" them in place.
Shear the DNA into small fragments (via sonication or enzymes).
Immunoprecipitate using an antibody specific to the protein of interest — this pulls down the protein + bound DNA.
Reverse crosslinks to separate protein from DNA.
Purify the DNA and sequence it (using next-gen sequencing).
Map the reads to the genome to see where your protein was bound.
42
What are the three types of super resolution fluorescence microscopy?
- Photoactivatable Localization Microscopy (PALM)
- SIM
- STED
43
Explain PALM
The concept here is that you excite a few of the photoactivatable GFP molecules so that they will emit photons that can be put on a Gaussian curve. You can find the center of the Gaussian curve to beat the resolution limit. You take multiple images, only activating some GFPs at a time and you can use a computer to construct the overall image.
44
Explain SIM
Take different images of the sample having an illumination pattern that changes orientation on each photo. The illumination pattern is lighting up the fluorescence molecules. Overlay the photos to see where the interference is (where it overlaps) to determine where the molecules are and get a clear image
45
Explain STED
Use an excitation laser to go over the same and detect the fluorescent molecule. There is a zone of depletion around the excitation light though which allows for more precision.
46
What do you need to ensure that you correctly inserted GFP with your protein of interest?
That GFP is in the same reading frame as your protein of interest
47
What are the key features of GFP
1. Direct detection
2. Species independent
3. Long half life
4. Folds independently
5. Can be fixed and still fluoresces
6. Has no localization signal
48
What makes EGFP improved?
It is brighter, better folding for mammalian cells, and codon is better suited for humans
49
Photoactivatable Protein
Non-reversibly activating a protein
50
Photo-convertible Proteins
You can switch the protein on or off
51
Photo-switchable Proteins
You can switch the color of the fluorescence
52
What is the issue with culturing B cells? What is the solution
They cannot be grown in a large population in cell culture because they die out. The solution is creating hybridoma cells. These are cells that are a fusion of immortalized cancer cells (myelomas) and B cells
53
What is the diffraction limited for electron microscopy?
0.1 nm
54
Do the samples for TEM have to be thick or thin?
They must be thin
55
What is one similarity between TEM and SEM?
They both must be performed in a vacuum
56
What is negative stain EM good for? How can you tell you're looking at it?
Negative stain EM is just good for small particles, the only issue is that membrane proteins do not show ultrastructure very well.
You can tell it is negative stain because you will see a very dark outline around your proteins of interest
57
Cryo-EM. What is it good for?
Very good for determining structures for large protein complexes. The sample is frozen in a small liquid and put on a specimen holder. The specimen holder is tilted as the electron beams are sent towards the sample, an image is collected each time and the specimen holder will rotate afterward.
The images are then collected and a 3D image is constructed
58
Immuno EM, draw it out and explain it
This technique is really good if you want to define where a protein is in a cell.
To visualize the antibody, it must be attached to a metal - like gold, which will come up as a dark spot. The gold is bound to protein A which binds to the Fc portion of antibodies
59
What does differential centrifugation rely on to separate organelles?
Size. The nuclei, the largest organelle, is pelletted out first
60
What are the two methods to breaking open the cell to expose the cell lysate? What are the negative effects of them?
The two methods to break open the cell are:
- Using a mild detergent
- Shearing such as sonication
The negatives of using a mild detergent is that it may also disrupt the membrane of organelles and rupture them. Similarly, shearing is too rough and ruptures the organelle
61
For equilibrium centrifugation, what could you use to generate the density gradient?
Sucrose or glycerol
62
True or false. Once already spun at its maximum speed, spinning twice as hard or for twice as long will lead to more separation.
False. Once you've already spun at max speed for several hours and ensured maximum separation, spinning harder and for longer will not help
63
For a Western blot, what are things the secondary antibody can have on it for read out purposes?
It can have an enzyme conjugated to it or a fluorophore
64
Does SDS denature proteins
Yes
65
Explain how immunoprecipitation or a pull down assay works?
1. Lyse your cells
2. Have your antibody bind to your proteins of interest
3. Add your beads which have protein A conjugated to them
4. Spin down the beads so you have your protein-bead complex
5. Wash
6. Separate your protein from the beads
7, Analyze
66
What is mass spectrometry good for?
Powerful tool for characterizing a protein, especially determining th emass of a protein. Tells information about what the protein is, what the amino acid sequence is
67
What do you need to maintain primary cells in culture?
1. Cells need to have a certain density --> contact inhibition
2. Cells require nutrients (salts, sugars, amino acids, vitamins, fatty acids)
3. Cells require proteins --> serum dependence
4. Cell growth depends on cell type
5. Cells need to grow from a surface --> anchorage dependence
5. Cells have a finite life span --> mortality
68
What are primary cell lines?
Cells taken directly from tissue
69
Senescence
Cells will initially grow and divide, eventually they reach a point where they can no longer grow and will just die
70
Cell line definition
After many cells reach a point of senescence in the cell culture, some of them are said to oncogenically transform and continue to divide despite initially having a finite amount of divisions. These cells now have the ability to grow and divide forever. Cells that can grow and divide forever are called cell lines
71
What are the different faces of the phospholipid bilayer?
The exoplasmic leaflet
The cytosolic leaflet
72
What are the three classes of lipids in the biomembrane?
- Phospholipids
- Sphingolipids
- Sterols
73
What head group do sphingomyelins have?
Phosphocholine head groups
74
Fluid mosaic model
The idea that individual lipids move fluidity in a 2D model. They eithe rspin or move past one another
75
What is usually found on the inside (cytoplasmic leaflet) of the plasma membrane?
PE, PS, PI (there's a very small amount of PI in general)
76
What is usually found on the outside (exoplasmic leaflet) of the plasma membrane?
All glycolipids, SM, PC
77
How is cholesterol divided on the asymmetrical bilayer?
Cholesterol is evenly divided
78
Which phospholipids have charged head groups? What does the cell do to compensate for these charges?
PS and PI have negatively charged headgroups, leading to the cytoplasmic domain to be very negatively charged. To compensate, many membrane proteins will have positively charged residues such as Lys and Arg on the side that faces the cytosolic part of the bilayer
79
How are phospholipases used as a tool to study the abundance of phospholipids. What is a limitation
You can add then and see how many phospholipids get cleaved essentially to determine what the relative abundance of them is.
However, they cannot cross the plasma membrane, so hydrolysis and cleavage can only occur to head groups that are on the exoplasmic domain
80
Which head group causes curvature?
PE has a small head group but long tails, this causes it to curve. PS has long tails and a large head, so it creates perfect cylindrical shapes
It is thought that PE is found in membranes that curve a lot like vesicles
81
Does adding cholesterol to sphingomyelins increase their stiffness?
No, sphingomyelins by themselves have such long tails that they are very stiff. Adding cholesterol would not help
82
What are all the different things that could affect the fluidity of the membrane?
1. Long vs short tail
2. Double bond (kink) vs no kink
3. Temperature
4. Presence of cholesterol
83
Integral membrane protein
Protein that spans the lipid bilayer. It has a hydrophilic side on both ends and an hydrophobic region for the part that passes the membrane
84
Lipid linked protein
Protein that is covaletntly linked to a lipid that is embedded in the plasma membrane
85
Peripheral membrane proteins
Directly or indirectly linked to lipid linked proteins or integral membrane proteins. The easiest to strip away with detergents
86
Explain Acylation, its variation, and which leaflet its located on
Proteins are bound to a lipid via a glycine residue at their N terminus. Sometimes, extra reinforcement can be added and the protein can also be linked at the cysteine residue
Located on the cytosolic leaflet
87
Explain Prenylation and which leaflets its located on
Proteins have their C terminal cysteine bound to a lipid
Located in the cytosolic leaflet
88
Explain GPI anchors
Hydrocarbon anchor that is used and is found on the exoplasmic side
89
What are the ionic detergents?
Sodium deoxycholate
SDS
90
What are the non-ionic detergents?
Triton-X-100
Octylglucoside
91
Draw the CMC graph for micelles and explain why monomers plateau
They plateau because there will always need to be a minimum amount of monomers in solution and once the micelles have been formed, that number of monomers have to remain the same to preserve balance
92
Can you explain what detergents to do integral membrane proteins above and below the cmc
Below the cmc: The detergent can insert themselves around the proteins and prevent them from sticking to other things, making teh membrane more leaky
Above the cmc: The detergents and phospholipids form a mixed micelle and can remove the integral membrane protein from the phospholipid bilayer, essentially dissolving it
93
Let's say you just performed a FRAP experiment and noticed that the highest your graph got after bleaching was 50%. What does this mean for the movement of your lipids/proteins?
They are 50% mobile
94
What are 4 reasons why lateral mobility could be restricted?
1. Membrane protein to ECM interaction
2. Membrane protein to cytoskeleton interaction
3. Membrane proteins interacting with neighboring proteins so tightly
4. Membrane proteins interacting with neighboring membrane proteins on another cell
95
What's an example of how lateral movement is restricted in epithelial cells?
Tight junctions between neighboring cells prevent the movement of proteins or lipids from the apical side to the basal side and vice versa
96
Draw out the permeability across a bilayer graph
97
Do symporters and antiporters use energy?
Yes, both of them do. And the molecules cannot move independently, they must always be cotransported together
98
Examples of a uniporter
GLUT1, the glucose transporter. Like all uniporters, the direction is reversible and it is specific one one molecule
99
What is Km a measure of, especially in the glucose example
Km is the concentration at 1/2 of Vmax. Vmax is dependent on the number of uniporters that there are. Km tells you the affinity a receptor has for its ligand, therefore the lower the Km, the higher the affinity the receptor has for its ligand
100
Symporter example, Na+ Glucose channel
Na+ moves down its concentration gradient and glucose will use that energy to move in the same direction (downward), even though its against its concentration gradient
101
What are ABC proteins?
They are proteins that liver, intestines,
and kidneys—sites where natural toxic and waste products are removed from the body. They use the power of ATP hydrolysis to do so
102
What is the order of the transcellular transport?
1. Na+ K+ Pump
2. Na+ glucose symporter
3. Glucose uniporter
103
What is the main driver of the membrane potential?
The leaky, nongated K+ channels
104
How does the action potential work
The cell is initally polarized (negatively charged), as Na+ wants to come in but cannot. There is only the leaky K+ channel
Then, an event depolarizes the cell (makes it slightly more positive), allowing for the opening of the Na+ channels and a rush of more positive charges (depolarized)
105
Without ATP, does the G-actin globular monomer stay stable?
No, it doesn't
106
What induces the polymerization of G-actin into F-actin
The presence of cations like Na2+ Mg2+ K+ in solution
107
How does the ATP binding cleft differ on the (+) end versus the (-) end
The ATP binding cleft on the plus end is facing the neighboring protein whereas on the minus end it faces freely into the solution.
108
What are the steps for actin polymerization?
1. Nucleation
2. Elongation
3. Steady State
109
Can you explain the steady state phase in actin polymerization?
The steady state phase is the phase when there is no net change in the number of actin filaments being added. You have an equal amount of filaments being added as ones that are being removed
110
At steady state, what concentration are the G-actin monomers at?
They remain at the critical concentration
111
What is the rate of polymerization dependent on for treadmilling?
The amount of free G-actin
112
Explain treadmilling. Then explain the asymmetry of the F-actin
Treadmilling occurs when the concentration of free G-actin is in between the critical concentrations of both ends. The (+) end has a very low Cc but the (-) end is slightly higher.
The plus end has ATP-G-actin, followed by ADP + Pi, and then ADP G actin, which dissociates readily from the structure
113
Phalloidin
Drug that binds to F-actin filaments (the interface between them) to stop depolymerization
Also a popular staining reagent for F-actin
*Not great because cells need to turnover their actin
114
Cytocholasin
Drug that binds to the (+) end and stops the addition of G-actin
115
Latrunculin
Binds to and sequesters ATP G-actin
116
Profilin
Profilin binds to free ADP-G-actin, opening up its cleft so the ADP can leave and the ATP can join. The profilin will then bring it to the (+) end (the area that profilin binds to wouldn't allow for it to be added to the (-) ends and it dissociates afterwards
117
Cofilin
Binds to the ADP F-actin to promote its disassembly and allow for free ADP-G-actins in teh solution
118
Thymosin B4
Acts as a buffer. Will bind to ATP-G-Actin when there is a lot is too much in the environment and dissociates when its lowered and needs to be used to grow the filament. Remember, in the cell, the concentration of G-actin is much higher than the Cc, so the cell must regulate when it wants to polymerize or not so that thymosin B4 can let go
119
CapZ
CapZ binds to the plus end of the filament and prevents growth or dissociation. Many regulatory proteins try to bind to the plus end first to prevent CapZ from binding
120
Tropomodulin
Tropomodulin binds to the minus end of the filament to prevent growth or dissociation. It is highly prevalent in muscle cells or RBCs.
Works with tropomyosin to stabilize
121
Gelsolin
When there is Ca2+ in the environment, Gelsolin can bind to the filament and break it - capping the (+) end and creating a new minus ends. When filaments crosslink, they create gels. But by severing these links, Gelsolin turns the filaments into a liquid again
122
Explain how branched filament assembly works
You have two NPF that has two domains (lets say its WCA) a WH2 and acidic domain. Each WH2 domain will bind to one G-actin. The acidic domain will bind to the Arp 2/3 complex. The ARp2/3 complex will then bind to an actin filament and addition of G-actin will continue. NPF will dissociate and you will hav a branched filament 70 degrees from the first
123
What are the proteins involved in Listeria mobility (host proteins) and what is the one protein Listeria uses. Explain how they come together to help Listeria
Host proteins:
Arp 2/3
ATP G actin
Cofilin
CapZ
Listeria protein: ActA
Listeria uses ActA which acts as a NPF. Therefore, its able to allow for the polymerization at its behind to push itself forward. CapZ is important for stability and Cofilin is to recycle ADP G-actin at the ends of the filament as the Listeria is pushed forward
124
For actin polymerization and use of the Arp2/3 complex in endocytosis, which NPF is used?
*Also used in phagocytosis
WASP
125
What are the two ways actin filaments can be linked
- Actin filaments can be parallel crosslinked (bundling)
- Actin filaments can be crosslinked (gel)
126
What do proteins need to link filaments?
Two F-actin binding sites, whether the protein has two on its own or needs to dimerize
127
The role of spectrin and the type of F-actin associated with it.
Spectrin forms a gel like meshwork right under the plasma membrane of RBCs. They are associated with short F-actins
128
Examples of two actin binding proteins
Spectrin -- crosslinkining
Ezrin --- parallel bundling
129
The role of Ezrin
To laterally bundle actin filaments relative to the plasma membrane
130
What is S1 of myosin?
The globular head domain that contains the actin binding site and the light chains
The ATPase activity lies in the S1 fragment. This activity increases when it is bound to an actin filamentq
131
Draw the basic structure of the different myosins, explain their key features such as function and step size
Myosin I Features:
- Single headed
- Variable number of light chains
- No heavy chains
Function:
Helps bring in vesicles during endocytosis
Step size: 10-14 nm
Myosin II Features:
- Double headed
- 2 of each light chain per head group
- Arranges in a bipolar way
Function:
Muscle contraction
Step Size: 8 nm
Myosin V Features:
- Long neck, 6 light chains
- Has a vesicle binding domain at its tail
Function: Vesicle transport
Step size: 36 nm
132
Briefly explain how the sliding assay works
Myosin I heads are tethered to a coverslip and there is fluorescently labeled actin that is close to it. The myosin will pull on the (+) end and push towards the minus end of the actin filament
133
For the optical trap, what does it determine about a motor proteiin?
- Its distance, force, and duration of movement
Myosin I heads are put onto a bead. An actin filament between two lights are lowered down to the bead so that one Myosin can bind to it and induce force.
134
Is the duty ratio of Myosin V high or low
The duty ratio is really high, this means that the globular heads slow down the release of ADP from their S1 which allows them to stay in contact with the actin for longer. A duty ratio above 50% means that it will be processive
135
Draw the packing of myosin and actin in the sarcomere
Also the sarcomere contracts about 70%
136
For muscle contractions, which molecules are required for the contraction (not myosin or actin)
ATP and Ca2+
137
Relationship between tropomyosin, troponin and contraction (thin filament regulation)
Tropomyosin is a rope life structure that has Ca2+ binding sites. It covers up the myosin binding sites on troponin. However, when there is an increase in Ca2+, tropomyosin moves and allows the active binding sites (myosin can now bind the the actin filament and have contraction)
138
Thick filament regulation
Typically, myosin in presented in a folded state. To have the myosin open , Ca2+ must bind to calmodulin which will then undergo a conformational change. Then, the conformational change allows for it to activate MLC kinase which can then phosphorylate the myosin. When the Ca2+ levels get low, the MLC phosphatase takes over and inactivates it.
139
Which myosin plays a role in mitosis?
Myosin II - the contractile ring
140
What is one of the effector proteins of Rho once it has been activated
Formin. Rho will bind to the RBD domain of formin, unfolding it to expose its FH1 and FH2 sites. Similar to how it was mentioned before, but the actin filament will grow from there. Additionally, FH1 will act as a landing site for profilin ATP G-Actin so it can be added on
141
Does Formin prevent CapZ from binding?
Yes
142
What is one of the effectors of Cdc42 once its been activated?
Activating WASP, the NPF, to encourage branching. It unfolds the WCA domains by binding to the RBD sequence, but it still requires PI(4,5)P2. This is called coincidence detection.
143
What are the different F-actins found in migrating cells?
- Branched actin found at the leading edge
- Stress fibers which are responsible for pulling the entire cell up after it releases its adhesion
- Contractile fibers which generate force to push the cell forward
144
Can alpha and beta tubulin both hold GTPs? Which GTP is hydrolyzed? Is it true that they are stable as a dimer?
Yes, they both hold GTPs, but the GTP in the alpha tubulin is covered by the Beta subunit, therefore, it never hydrolyzes. These two are very stable as a dimer, but they are not very stable as monomers
145
How many filaments are put together to create a microtubule? Is there polarity?
13 protofilaments
Yes, the end with the Beta subunit exposed is the (+) end and the end with the alpha subunit exposed is the (-) end
146
What is the temperature dependence of microtubules?
At low temperatures, the microtubule depolymerizes (4 degrees). When it's back to 37 degrees, it reassembles. This was helpful for experimentation
147
What is it called when a growing microtubule catastrophically disassembles? Can you draw an example of what this graph would look like and label it
Dynamic instability. This is when the growing microtubule suddenly disassembles. However, it can be rescued and can assemble again.
148
True or false: The addition of a dimer to the (+) end on a growing microtubule enhances the hydrolysis of the GTP in the formerly terminal Beta subunit to GDP and Pi
True. The inorganic phosphate is then released to yield a microtubule with predominantly GDP-β-tubulin down its length.
149
Explain the mechanism between going from a growing end to a catastrophe to a rescue
When GTP is bound to the filament end, it is only slightly curved. When you take into consideration the lateral interaction with GTP cap protofilaments, its able to keep the microtubule together. But remember, the GTP Beta tubulin really only exist in the cap, along the length of the microtubule, the GTP is converted into GDP + Pi and then GDP. The energy is stored inside the microtubule. When the GTP cap is lost, the filaments want to assume a rams horn position, but because there was so much energy in the microtubule, it will QUICKLY peel back due to "work". Rescue can occur because there are little islands of GTP tubulin, these can cause the reassembly of the microtubule
150
Taxol
Drug that prevents the depolymerization of the microtubule (something like this could prevent mitosis)
151
Colchicine
A drug that prevents polymerization of microtubules and sequesters GTP tubulin dimers
152
Nocodazole
A drug that also prevents polymerization of the microtubule
153
What are the various MTOCs in cells
Centrosome (interphase)
Spindle poles (mitosis)
Basal bodies
154
What are the functions of MAPs, what are their structure?
MAPs are important for the stability and spacing of the microtubule (increases growth rate and reduces catastrophe)
Has two domains. One is positively charged to bind to the negatively charged microtubule. And then the other domain comes out in a right angle
155
What is the function of +TIPs, what is required for them to associate with the microtubule?
The microtubule must be growing and on the plus end. +TIPs work to stabilize the microtubule, increasing growth and suppressing catastrophe
156
What is the general structure for kinesin I
Head domains followed by linkers followed by stalk of heavy chain domains followed by tail domain that is associated with the light chains
157
What are the different main kinesins?
Kinesin I: important for organelle transport
Kinesin 5: Pushes apart interpolar microtubules during mitosis. Bipolar head
Kinesin 13: only the head and linker. Important for depolymerizing kinetochore microtubule during mitosis
158
Rarer kinesin, 2 and 14
Kinesin 2 is also for organelle transport but is heterotrimeric
Kinesin 14 is the only kinesin to walk in the minus direction
159
Explain the role of dynactin
Dynactin has two domains that have different roles
Domain 1: This domain is based around a short actin filament. This is attached to the cargo. Around the actin filament are Arp1 proteins. At the (+) end is a CapZ protein
Domain 2: p150 Glued reattached dynein after a cycle because it isn't processive
160
What is the role of dynamitin
To connect the two domains of dynactin
161
Is a secretory vesicle going to the Golgi using dynein-dynactin or kinesin I
Dynein-dynactin
162
Is mitochondria using dynein-dynactin or kinesin I
Both
163
Are pigment granules using dynein-dynactin or kinesin
Both
164
Is going to the late endosome or the lysosome dynein-dynactin or kinesin?
Dynein dynactin
165
The role of pigment granule transport in skin color change
There are certain cells that have pigment granules in them. If you have a high amount of cAMP, the granules are distributed via kinesin 2. If you don't have a high amount of cAMP, dynein dynactin aggregates them towards the center (causing a pale look)
166
Role of kinesin and dynein in axonal transport
Axons are always releasing materials like proteins at their axonal terminal. But they need to be replenished. Most materials are made in the cell body and must be carried down by kinesin I. These kinesin 1s will bind to the same organelle/cargo as dyneins to be carried back
*it isn't known how one motor protein is switched off while the other one is on
167
Which one is larger, cilia or flagella?
Flagella
168
What is the structure of a flagellum?
9 + 2 axoneme
169
Nexin
Proteins that joins the doublets in teh 9 +2 axoneme
170
What causes the bending of cilia and flagella?
The dynein dynactin interacting B tubule.
171
Role of kinesin and cytoplasmic dynein in IFT
Kinesin II and Cytoplasmic (as opposed to axonemal dynein) walk on the outer part of the double (between that and the plasma membrane to move things in the same direction that they typically do)
172
What occurs during interphase?
- DNA duplication
- Centrosome duplication
- Cohesin rings are attached
173
What is the role of the astral microtubules? kinetochore? interpolar?
- Astral: They interact with the periphery of the cell to ensure that the spindle poles are properly in position
- Kinetochore: Will cause the separation of the sister chromatids
- Interpolar: Pushed the duplicated centrosomes apart during prophase and push the spindle poles apart during anaphase B
174
What do kinetochores attach to? (two answers because its sandwiched)
Centromeric DNA and the + ends of microtubules
175
During prophase, which motor proteins facilitate the separation of the centromeres? This is also done in anaphase B
Kinesin 5 and Dynein
176
They link the plus ends of astral
microtubules to components of the actin cytoskeleton at the cell cortex, for example; by
moving toward the minus end of the microtubules, the dynein motors pull the spindle
poles toward the cell cortex and away from each other.
177
Which kinesins are associated with the chromosome arms?
Kinesin 4, 10
They hold on to the chromosome arm like cargo and move towards the plus end
178
What are the two important steps in prometaphase?
Bi-orientation (this just means that you have a microtubule on each side of the sister chromatids)
Congression (the back and forth oscillation to ensure the chromatids are assembled on the metaphase plate)
179
Role of kinesin 7 in prometaphase (and also in congression)
Helps to make other chromosomes gain bi-directional attachment
During congression, it holds onto the growing end so that tubulin dimers can add on
180
Role of dynein in prometaphase
First attaches to one kinetochore and pulls it really close to the spindle poles so that another microtubule could reach over and it could gain bi-directionality
181
Role of Kinesin 13 in prometaphase
Important for depolarization during the oscillation that occurs in congression. Dynein also helps to pull it back
182
Explain the FRAP experiment when they were looking at anaphase A
They bleached one area of the microtubules and observed how the bleached area got moved backwards but only at the plus ends, it wouldn't get pulled back very much at the minus ends because there is no depolymerization there
183
Are intermediate filaments heterlogous?
Yes, there are so many different kinds of intermediate filaments
184
Are intermediate filaments stable, and strong?
They are both, these are separate properties but they are both true.
185
Do intermediate filaments have intrinsic polarity and motor proteins?
No
186
Are intermediate filaments only found in animals?
Yes and one class of insects
187
What is the role of plectin in intermediate filaments
Helps them bundl and associate with one another
188
Connective tissue
This is bone and tendon made out of extracellular matrix protein
189
Homotypic vs heterotypic interactions
This is when CAMs (cell adhesion molecules) adhere to one another or do not adhere to one another. This is different from their binding partner which would constitute heterophilic vs homophilic interactions
Homotypic is the same CELLS interacting with one another
190
What microfilament is integral for adherens junctions? What type of CAM mediates the connection between adherens junctions
Actin filaments
Cadherins mediate the connection between adherens junctions
191
What other junctions are cadherins predominantly found in ?
Desmosomes
192
Cadherins, are they homotypic or heterotypic? Homophilic or heterophillic?
Cadherins are both homotypic and homophilic
193
Explain the difference between cis and trans interactions with cadherins
Draw this out
194
Are cadherins Ca2+ dependent?
yes
195
For cis interactions, which domains interact? What about trans
EC1 and EC2
EC1 and EC1
196
What filaments are attached to desmosomes?
Intermediate filaments
197
What are the structure of gap junctions?
You have 6 transmembrane connexins and form a connexon
198
Name ECM proteins
- Fibronectin
- Type IV collagen
- Laminins
199
What shape are laminins
Cross shaped
200
What is the structure of Type IV collagen
Triple helix structure, this can only form because glycine is small and they have a repeating sequence of Gly-X-Y. It is really flexible
201
GAGs and hylauronan
GAGs are a type of proteoglycan that are very negatively charged. One type is hyaluronan which is important for hydrating, giving turgor pressure, and compression on joints
202
What are the morphological steps for cell death?
1. Chromatin compaction, Condensation of Cytoplasm
2. Breakdown of nuclear envelope, Nuclear fragmentation, Blebbing, Cell fragmentation
3. Phagocytosis
203
What happens to the mitochondria during apoptosis
Undergoes fission and loses its membrane potential
204
What happens to the nuclei during apoptosis
Nuclei condensation
205
What happens to the DNA during apoptosis
Fragmented by nucleases
206
Ways to assess apoptosis for an individual cell:
1. Morphology: nuclear condensation and fragmentation, plasma membrane experiences blebbing
2. DNA staining to see if DNA is fragmented. During apoptosis, you have more free DNA ends
3. Staining for the protein, caspase
4. PS on the exoplasmic side of the plasma membrane
207
Ways to assess apoptosis for a cell population:
1. DNA fragmentation
2. Look for the cleavage of specific substrates that are caspsase targets such as PARP, poly-ADP, ribose polymerase)
208
What are ced mutants and why they were important for the study of apoptosis
Ced mutants were discovered in C. elegans. In the development of a hermaphrodite, 131 somatic cells died. They wondered why and induced mutations to discover that there were genes that were set to program death and for proper phagocytosis, when they are mutated, these process are stopped . These were known as the ced mutants
209
CED3
Protease (caspase in mammals) that cleaves key cellular proteins - inducing apoptosis
210
CED4 (Aparf-1)
This is a protease activating factor. It will activated CED3
211
CED9
This is localized on the outer mitochondria. It will bind to CED4 (forms a complex with one of the dimers), inhibiting it from activating CED3
212
How do Caspases function
They contain a key cysteine residue their catalytic site and uses the cysteine as a knife to cut right after the Aspartate (towards the C-terminal)
213
How is mammalian caspases different from CED3
In mammalian cells, you have a cascade of caspases that amplify the death signal
214
How are caspases contained until it's time for them to be used?
They are all made as procaspases that require the binding or a protein complex or proteolytic cleavage to activate
215
Which proteins are the sensors of the apoptotic pathway?
Bcl-2 and CED-9. They are transmembrane proteins that are localized to the outer mitochondrial membrane
216
What is the analog of CED9 in vertebrates
Bcl-2
217
What is the only domain that Egl-1 has?
BH3 domain
218
What is the role of all members of the Bcl-2 family?
They either are anti-apoptotic (preserve the integrity of the outer mitochondrial membrane) or they are pro-apoptotic (increase the permeability of the membrane)
219
Do all Bcl-2 family proteins reside on the outer membrane of the mitochondria?
No, some translocate to the inside
220
Is Bcl-2 pro-apoptotic or anti-apoptotic? What is its domain
Anti-apoptotic
BH4 BH3 BH1 BH2 TM
221
Is Bak pro-apoptotic or anti-apoptotic? What is its domain?
BH3 BH1 BH2 TM
pro-apoptotic
*Bax is the same
222
Explain the process of how cell death is activated in full, it helps to draw this out.
1. BH3 only domain protein, Bad, will bind to Bcl-2, triggering the separation of Bak/Bax from it
2. Bak/Bax will form pores in the outer mitochondrial membrane (Bax is mainly found in the cytosol for healthy cells but it can come and also make pores)
3. Cytochrome C escapes through the pores
4. Cytochrome C binds to Aparf-1 which turns the inactive Caspase 9 to an active Caspase 9
5. The active Caspase 9 cleaves the pro-caspase 3 into active caspase 3
6. Active caspase 3 will use its cysteine residue to cut its target proteins
223
What is the role of BH3 only domain members of the Bcl-2 family?
To regulate the activity of Bcl-2
224
What is observed with PS during apoptosis? What is its function
PS is present on the exoplasmic face of the phospholipid. They act as an "eat me" signal and bind to PS receptors on neighboring cells so they can be phagocytosed
225
If i wanted to visualize or know if a cell was going through apoptosis through binding to PS, what should I use?
Annexin A
226
How is it that PS ends up on the exoplasmic face? Use flippases and scramblases in your answer
Typically, flippases use the power of ATP to move phospholipids to their correct leaflet, even though scramblases move them to reach equilibrium. When caspase 3 is activated, it will cleave both the flippase proteins (inactivating them) and cleave one of the scramblases (activating it). The scramblase will dimerize with two other proteins to create a translocase and move ALL phospholipids, including PS, to the exoplasmic face
227
What was the rationale of the experiment with the neurons and the chicken limbs
Scientists knew that the body overproduced neurons and that only the neurons who made successful connections with tissue got the chance to survive. The other ones went through apoptosis. They wanted to know what regulated the apoptosis
228
What were the conclusions of the experiment with the neurons and the chicken limbs
Removed limb buds (the target tissue for motor neurons):
Result: Fewer motor neurons survived in the spinal cord.
Grafted extra limb buds (added more target tissue):
Result: More motor neurons survived in the spinal cord.
Target tissues (like limb buds) release survival factors, often called neurotrophic factors (e.g., NGF – Nerve Growth Factor).
Neurons compete for these limited survival signals.
Only neurons that make functional connections receive enough survival factor to avoid apoptosis.
Apoptosis is a normal and necessary part of nervous system development to match the number of neurons to the size/needs of the target tissue.
229
What do neurotrophins bind to?
Trks - a type of RTK
230
What is the function of neurotrophins?
To provide survival signals to different nerve cells
231
True or false: Each neurotrophin binds with high affinity to ONE type of Trk
True. only one type of Trk
232
What experiment did scientist do to discover that neurotrophins are required for neuronal survival
They did a knockout of a neurotrophin or its receptor (remember only one neurotrophin can bind to one type of receptor). When they did this for the different pairs, there was no neuronal survival
233
How do neurotrophins prevent cell death? What is the BH3 domain protein?
Many of them activate the PI3K pathway. Binding of a neurotrophin activates PI3K which activates PKB. PKB will phosphorylate Bad, preventing it from interacting with Bcl-2
Bad
234
How is apoptosis induced as a response to damaged DNA? What is the BH3 domain protein?
p53 activates the BH3 domain protein PUMA. Puma binds to Bcl-2, separating it from Bak/Bax and then this pathway will lead to apoptosis
235
How is apoptosis induced as a response to loss of integrin signaling? What is the BH3 domain protein?
When the cell is separated from its substratum (its no longer connected to the ECM), the BH3 domain, Bim, will bind to Bcl-2 and cause the apoptotic pathway
236
Can TNFalpha work by remaining on the surface of macrophages and other immune cells?
Yes, it could be secreted or remain on the surface
237
What are the two death signals
TNFa and Fas ligand
238
What is necroptosis
Cell death as induced by extracellular signals
239
Explain how necroptosis works with TNFa as an example
TNFa will bind to the extracellular domain of a death receptor. The intracellular death domain will associate with two other proteins. These proteins will recruit and activate Caspase 8. Caspase 8 will cleave and activate Caspase 3, 6, 7
Caspase 8 will also cleave Bid into just its BH3 domain tBid. This will bind to Bcl-2 and allow Bak/Bax to leave
240
Re-explain the part of Caspase 8 and Bid
Caspase 8 cuts Bid into just its BH3 domain known as tBid. This will bind to the Bcl-2 and allow for the dissociation of Bak/Bax
241
Do Caspase 9 and Caspase 8 have the same targets
Yea, caspase 3,6,7
242
What kind of motif is the EF hand family
Helix loop helix
243
Does Calmodulin activate all the proteins it binds to?
No, it can activate or deactivate them
244
What is one enzyme that is activated by calmodulin that is mediated by a different GPCR pathway?
cAMP PDE
245
What pathway leads to the release of Ca2+
The Phospholipase C pathway
246
What pathways work together to activate GP in muscle cells?
The Adenylyl Cyclase Pathway and ?Phospholipase C (mainly just Ca2+)
247
What binds to the muscarinic receptor
Acetylcholine
248
What kind of Ga protein is associated with the muscarinic receptor? Does Gby or Ga bind to the K+ channel
Gai
Gby associates
249
What does the opening of the K+ channel do?
Causes hyperpolarization and reduces muscle contraction
250
4 ways to activate transcription factors
1. Classic GPCR binding to G protein and activating TF down the line
2. RTK activating the TF
3. Binding of ligand leads to the separation of a multiprotein complex, allowing for the release of the TF
4. Binding of ligand prevents the inhibition of the TF's activity
251
What are 4 pathways RTKs take?
1. Activation of Ras which leads to the activation of MAPK
2. Activation of Phospholipase C which leads to Ca2+
3. Activation of PI3K which leads to PKB
4. Activation of JAK which activates STAT6
252
What does ligand binding do to RTKs
Forces it to become a dimer
253
Is there a lot of tyrosine phosphorylation in the cell? What is the main role of p-Tyrosine?
No there isn't. It's usually used as a signal in growth control
254
What about the dimerization of the RTK monomers allows for the kinases to phosphorylate eachother despite the presence of the activation loops?
They are close together
255
What molecule binds to the RTK for Ras activation
FGF
256
Ways in which to attenuate the signal
1. Receptor is recycled, ligand is digested
2. Receptor and ligand are digested
3. Receptor is phosphorylated - inactivated as a result
4. G-protein is phosphorylated - inactivated as a result
5. The signal from the receptor to teh G protein is inhibited
257
What happens during G1 phase:
- Growth of cell
- Preparation of RNA and proteins for the cell cycle (s phase)
258
How can you infer what part of the cell cycle budding yeast is in?
by the bud size
259
How can you infer what part of the cell cycle fission yeast is in?
By the length of the cell
260
What are the different CDKs?
G1: G1 and G1/S CDK
S: S CDK
Late G2: M CDK
261
What is the role of SCF in the cell cycle?
It is a ubiquitin ligase that allows for the movement from G1 to S phase
262
Draw out the graph that shows all the CDKs, their associated cyclins, and any other proteins during the cell cycle
263
What allows for the sharp activation or inactivation of the cyclin CDK complex
In part due to the periodic degradation and synthesis of cyclin
264
How to remember the names and order of the cyclins
DEAB
265
What is the role of SCF and APC/C
They are used to degrade cyclin (via ubiquitination) to restrict the cyclins to the correct phase of the cell cycle. This drives the cell cycle forward and prevents it from going backwards
266
What amino acid is the ubiquitin added to for polyubiquitination (and eventual destruction via the proteasome)
It is added to the LYSINE
267
Are there CDKs or cyclins expressed in G0
No
268
What leads to early response genes? How are delayed response genes created?
Addition of growth factors leads to early response genes. These genes become TFs that bind to the regulatory sites on delayed response genes to then activate them
269
Is Cyclin D an early response or delayed response gene?
Delayed
270
Can you explain the experiment with Cyclin D and BrdU
This experiment aimed to recognize the role of Cyclin D in progressing the cell cycle past the restriction point
1. Cells had growth factor added to them
2. Anti-Cyclin D antibodies were given to one group of cells
3. BrdU was added. This is a substitute for Thymine so that we can track if DNA synthesis had occured, aka if the cell was able to move into S phase
4. BrdU was detected in the first group but not in the one where Cyclin D activity was blocked
271
What does Cyclin D actually do to progress the cell cycle forward?
Cyclin D will phosphorylate Rb sos that it stops inactivating E2F. Active E2F can transcribe cyclin E and A which are important for G1/S and S phase. Cyclin E continues to phosphorylate Rb
272
What are other things that can prevent CDK activation even with present cyclin?
- Inhibitory phosphates
- Inhibitory proteins called CKIs
273
*Look at notes on Lec 23 to see this all come together. But try to explain this process from the beginning
274
What are the two things APC/C does when activated by Cyclin D CDK
1. It causes the degradation of M cyclin
2. It degrades securin from separase
275
What is one important function of MCDK as it relates to the nucleus. What proteins to they phosphorylate
Mitotic CDK leads the the breakdown of the nuclear envelope
1. They phosphorylate the nuclear lamins (PKC also does this)
2. They phosphorylate nucleoporins
3.
276
What happens when MCDK phosphorylates the integral membrane proteins of inner nuclear membrane?
The proteins lose their affinity to chromatin
277
How does the formation of the nuclear envelope work?
- The integral membrane proteins get dephosphorylates and they can bind to chromatin again
- The ER creates outward protrusions outward that come to form the double membrane of the nucleus
- Nucleoporins can associate with the NPC again
278
Where does the nuclear membrane retract during this?
Into the endoplasmic reticulum
279
What are the three very general things that every checkpoint needs
- Sensors
- Signaling cascade
- Effector
280
Are there checkpoints for DNA damage?
Yes
281
Is there a checkpoint for kinetochore assembly?
Yes
282
Is there a checkpoint for Spindle assembly?
Yes
283
Is there a checkpoint for growth?
Yes
284
Is there a checkpoint for DNA replication?
Yes
285
Draw out the diagram about cell cycles and checkpoints (DNA-PK)
286
What does serum contain in it?
Growth factors
287
What are two things that cancer cells do to evade needing serum?
1. Produce their own serum (autocrine)
2. Upregulate their growth pathways
288
What is the visual difference between fibroblast and cancerous fibroblast?
1. They are supposed to look flat and 2D, they are 3D and round
2. They are supposed to be in a monolayer not on top of one another
3. Should've exited the cell cycle but they remain in it
289
Cell lineage
The development of cells under a pattern of cell divisions
290
For cells that undergo asymmetric cell division, what can they vary in?
Shape, size, and proteins
291
What is required for the parent cell to do before asymmetric cell division
This requires for the parent cell itself to become asymmetric or polarized before it divides so it can distribute unequally to its daughters
292
In some cases of asymmetric cell division, is it true that one of the daughter cells can be exactly like the parent and the other one is a different cell?
yes
293
Stem Cell
An unspecialized cell that can reproduce and turn. into a more specialized cell
294
What stage/type of stem cell are all adult stem cells? What does this mean?
They are all multipotent stem cells. This means they can specialize into many different cell types but not all of them. It has restricted potential
295
What type of categorization for stage do zygotes fall into? Are zygotes considered stem cells?
Zygotes can give rise to any type of cell, therefore, they are truly considered to be totipotent. However, zygotes cannot divide, so they technically aren't stem cells
296
iPs
By giving a fully differentiated cells just a few TFs, they are indistinguishable from ES cells
297
What are two ways to classify stem cells
- Based on their potential
- From where they originate
298
What kinds of cells/what stage is an organism totipotent?
From being a zygote to the 8 cell stage
299
What kinds of cells/what stage is an organism multipotent?
Blood, intestinal cells, hair follicle stem cells
300
Are there tissue stem cells for every tissue in the body?
No, the heart doesn't have stem cells
301
Role of stem cells
Responsible for tissue turnover and repair from injury
302
As time passes, what happens to its stem cells?
the numbers of certain stem cells often decreases as the
organism ages, limiting their ability to replace certain outworn cells or tissues. This can lead to aging
303
Are tissue stem cells programmed to divide frequently
No, overexpression of this can cause cancer
304
Unipotent
Only make one type of cell such as germ line stem cells which only make germ cells
305
Draw out and explain the location of the germ line stem cells in drosophila
- Drosophila have ovaries that have ovarioles that have germanium. Two types of stem cells exist in the germanium: somatic stem cells and germ line stem cells. At the tip, cap proteins release molecules like Dpp and Gbb to suppress Bam, which allows for cell differentiation. This prevents the cell from differentiating. When the stem cell divides, one cell remains close to the cap cells, ramining undifferentiated. However, the other one does not, which causes it to differentiate. This is similar for the somatic stem cells which give rise to things like eggshells
306
What do somatic stem cells give rise to?
Egg shell
307
What is the name of the location where stem cells are found in intestines?
Crypts
308
Can you explain the birth of stem cells, paneth cells, and how they differentiate?
Paneth cells release secreting signals to maintain stem cells. These stem cells, when they are no longer receiving signals, will turn into transient precursors, which can divide and move up the villi until they are fully differentiated.
309
What are the fingerlike regions of the epithelial cells called?
Villi
310
Explain the Cre-ER experiment they did with Lgr5. Draw it. This is called Lineage Tracing
Question: Are Lgr5+ cells at the base of the crypts really multipotent stem cells responsible for renewing the intestinal epithelium?
Because they noticed that differentiated cells also had the Lgr5 GPCR
Lgr5 promoter drives ER-Cre expression:
Only Lgr5+ cells produce ER-Cre recombinase (an engineered enzyme that stays inactive until induced).
ER-Cre is activated by an estrogen analog:
This ensures precise control: researchers can time when recombination happens by adding the analog.
Cre-lox system activates β-galactosidase:
Once Cre is active (in the nucleus), it removes a stop cassette that blocks a β-galactosidase (LacZ) reporter gene.
Now, that Lgr5+ cell—and all its descendants—permanently express LacZ, which can be stained blue.
Right after induction: Only Lgr5+ cells are blue → confirms that the label starts in true stem cells.
A few days later: Many different epithelial cells along the crypt-villus axis are blue → meaning all differentiated lineages came from the original Lgr5+ cells.
311
What is the process above called
Lineage tracing
312
At the 8 cell stage, all cells are ___ but when they reach the 16 cell state, some have committed to differentiation
Totipotent
313
Inner mast cells (do you know what this looks like)
Small group of cells that will give rise to all the tissues in the embryo. Blastocyst stage
314
Trophectoderm
This is the outer layer of the blastocyst. These cells do not become part of the embryo. Instead, they form extraembryonic structures, mainly the placenta — which supports the embryo during pregnancy.
315
What can pluripotent stem cells not turn into?
All the other inner mast cells are pluripotent. They can transform into any cell except for extraembryonic ones such as the placenta
316
What can you do to the inner mast cells to make them embryonic stem cells? What kind of stem cells are they based on potential?
If you culture the inner mast cells, they become embryonic stem cells over time. They maintain their pluripotency
317
Can you explain what Somatic-cell nuclear transfer (SCNT) is, what is the success rate like?
This is when you take the nucleus of a fully differentiated somatic cell and put it into an egg that has had its nucleus removed from it. This is then implanted into a foster mother. This can lead to the rise of a normal embryo
The success rate for this is extremely low and the animals have high frequency of diseases
318
So, is it true that differentiated cells can revert back to being totipotent? Explain the experiment with the GFP that showed this.
Yes.
Mouse olfactory neurons are not supposed to grow and divide. They were labeled with GFP and their nuclei was taken out and put into an egg. Some of the eggs formed blastocysts and the inner mast cells were cultured to develop ES cells. The ES stem cells were then inserted into tetraploid blastocysts (this blastocysts has 4 chromosomes, it can make extraembryonic components but not embryonic components, so if an embryo were derived it would completely be from the olfactory nuclei cells). This was successful, and every tissue/cell was green
319
What are the three master transcription factors for "stemness", what cells are these found in?
Oct4, Nanog, Sox2. These are in ES cells
320
What are the functions of these master transcription factors?
- Repress any genes that promote differentiation
- Activate genes that promote self-renewal and pluripotency
321
Nanog, Oct4, and Sox2 are present in the process of converting ICM into ES cells (resulting in pluripotency). Are any of the transcription factors used in any other stem cells?
Sox2 is also used to maintain multipotency in neuronal stem cells.
- Nanog and Oct4 are exclusively for creating pluripotency in ES
322
True or false: Nanog, Sox2, Oct4 TFs form an autoregulatory loop, inducing their own expression as well as that of others. They also bind to transcriptional control regions of protein coding genes and micro-RNAs
True
323
What transcription factors, when introduced into a fibroblasts could induce pluripotency?
NOT NANOG
OKSM factors:
Oct4
Klf4
Sox2
Myc
324
Can only fibroblasts become iPSc?
No, keratinocytes and other cells have been induced
325
Can iPSc cells create an embryo?
Yes, if they are cultured, they can be introduced into a blastocysts and induce the formation of all the embryonic cells including the germ cells
326
How come cells remain in iPSc state even after the OKSM factors are no longer being given?
The OSKM factors engage in a self-sustaining feedback loop and activate each other.
327
True or false: Myc overexpression leads to the speeding up of the reprogramming process
True
328
If reprogramming was successful, which gene is permanently ALWAYS expressed? (N)
Nanog
329
What are some major changes that differentiated cells make to become pluripotent (not the steps)?
- Chromatin remodeling to express genes needed for pluripotency
- Telomerase activity is activated
- Metabolic activity: originally uses highly respiratory methods which are characteristic of differentiated cells. They switch to doing glycolysis which is good for cells that are proliferating
330
OSKM factors induces a series of events over a period of days to
weeks.
Just look at the diagram
1. Downregulate somatic markers
2. Mesenchymal to epithelial cell (PAY ATTENTION)
3. Pluripotency genes activated
4. Telomerase genes activated
331
Why is C.elegans particularly good for a lineage study?
Every C.elegans organism has the exact same lineage "route" for the development of every cell type
They are also transparent, have fast life cycles, and are easy to induce mutations into
332
In C. elegans, are all of the early cell divisions symmetric or asymmetric?
Asymmetric
333
Jot down the chart for C. elegans early cell divisions
334
The P0 cell is clearly asymmetric. What is the name of the contents that's asymmetrically given to one cell? What is this for?
P granules. They're for the development of the posterior. They will always segregate to the side that will create germ line
335
After P0 divides, which daughter cell gets the P granules?
P1. AB is larger though
336
What is the function of p granules
Unknown. Thought to store mRNA
337
Do the mass of the cells get larger as the cells divide?
No, they stay the same size and split up in the initial amount of mass they had
338
What are the two Par complexes?
Par3-Par6-aPKC is in the anterior
Par1-Par2 is in the posterior
339
Par6 and Cdc42
Par6 recruits Cdc42 to maintain the functional polarity of the zygote. A Cdc42-GAP (which turns Cdc42 off) is in the posterior, keeping Cdc42 active only in the front.
340
How do the Par genes exclude each other
If Par3-Par6-aPKC is present, it kicks out Par2 and Par1, and vice versa. This is done through phosphorylation
341
Cells can acquire multiple mutations to develop cancers according to the mutli-hit hypothesis. What process do cancer cells go through to invade tissue?
Invadopodium
342
What are the three types of genes that could be mutated to induce cancers?
Proto-oncogenes (dominant)
Tumor suppressor genes (recessive loss)
Genome regulatory proteins (recessive loss)
343
What are the two types of mutations that could lead to these cancers
Small mutations: such as SNPs, insertions or deletions
Or gross chromosomal alterations
344
Explain hyperactive proteins made in normal amounts
345
Explain the different was c-onc can lead to cancer
1. Single mutation makes hyperactive protein
2. Single mutation in promoter allows for overexpression
3. Gene duplication leads to overexpression
4. Chromosomal rearrangement leads to fusion protein that is hyperactive
5. Chromosomal rearrangement introduces an enhancer to a gene
346
Can you explain how CML works via translocation
You can have translocation between chromosome 22 and 9, creating the philadelphia chromosome. This fuses together the genes ABL and BCR, creating a new protein kinase that can phosphorylate plenty of targets that ABL normally doesn't. Imatinib can be used to bind to its catalytic site and prevent it from activating anything else
347
Can you explain how Burkitt's Lymphoma arises
Again, you have a chromosomal translocation. But in this case, you have Myc, a gene that is only supposed to be transiently expressed, now next to the heavy chain of antibodies, something that is expressed often. This leads to a lot of Myc and Myc is important for starting G1 of the cell cycle
348
Retroviral oncogene example in RSV
c-Src is a normal proto-oncogene found in chicken and other species.
v-Src (from Rous Sarcoma Virus, RSV) is a mutated, viral version of c-Src that acts as an oncogene.
v-Src lacks the C-terminal tail (including Tyr527) that normally keeps c-Src inactive.
In c-Src, Tyr527 is phosphorylated by another kinase and binds to the SH2 domain, inhibiting c-Src's activity.
Because v-Src can’t be inhibited this way, it remains constitutively active, driving uncontrolled cell growth and cancer.
349
What is the other method in which viruses can cause cancer? Why is it slow?
If viruses integrate in front of the wrong gene, their LTR domains can act as enhancers that drive gene expression. This would be slow because you'd have to (out of bad luck) bind right before an oncogene. And since we know that cancer requires multiple hits, its unlikely to get multiple hits where your virus just so happens to be in front of an oncogene
350
Classic tumor suppressor genes:
p53
Rb
p16
351
Although tumor suppressor genes require a double knockout, they often appear...
Dominant in terms of a family tree
352
What are the three ways that you can have a loss of heterozygosity?
1. Unequal splitting of chromosomes during mitosis
2. Crossing over*
3. The non-mutated allele stops functioning
353
ATM to p53 pathway. Explain how p53 works
Typically, MDM2 degrades p53 when there are no signals. When there is danger, ATM will recognize it and phosphorylate p53 (stabilizing it) and MDM2 (destabilizing it). Stable p53 will then bind to genes to induce apoptosis, signal for cell repair, or just halting (p21)
354
An example of DNA Repair Mechanism: MSH
Mismatch repair (MMR) is a system that fixes mistakes made during DNA replication, like:
Mismatched bases (e.g., G paired with T)
Small insertions or deletions (e.g., one base accidentally skipped or added)
Works by cutting out section and waiting for DNA polymerase and DNA ligase. Uses DNA methylation to know the original one
355
Histone modifications and what they lead to:
- Acetylation: causes chromatin decondensation (on lysine)
- Methylation: causes chromatin compaction (lysine or arg)
- Phosphorylation: two phosphates are added introducing two negative charges (-OH)
- Ubiquitination: Added to H2A and H2B C terminal lysine
356
If you want genes in a high amount, are you going to use CpG island promoters?
No, you will most likely use a TATA box
357
What are the different domains of RNA polymerase?
RPB1 and RPB2
358
CTD sequence? is this required for viability?
Tyr-Ser-Pro-Thr-Ser-Pro-Ser
Yes, it is required for survival, at least 10 are required
359
Does the CTD start with it being phosphorylated? When does this happen?
No, once transcription has started and the polymerase has moved away from the promoter, the CTD's serine and some tyrosine get phosphorylated
Large chromosomal puffs show genes that are being actively transcribed
360
What are the two important transcription factors that we have to remember
TFIID and TFIIH
361
What does TFIID do?
It helps position the RNA polymerase due to its TATA box binding protein (TBP)
362
What does TFIIH do?
- Is a helicase that uses the power of ATP to unwind the DNA
- Has three subunits that come together to form a kinase that phosphorylate the CTD domain. This will now act as a binding site for proteins that add the 5' cap
363
Most transcription factors have at least two domains
1. DNA binding domain (n terminus)
2. Activation domain (c terminus)
364
Structure of lipophilic receptor
N terminus is the activation domain, then DNA binding, then at the C terminus is the hormone binding domain
365
Inverted repeats NR:
- Homodimers
- Translocated into the nucleus
- Because they are cytosolic
ex. glucocorticoid receptor
366
Direct repeats NR:
- Heterodimer with RXR
- Nuclear
- TA by switching from repressor to activators
Act as repressors by directing HDAC at nearby nucleosome . WHen they undergo their conformational change, they bind to HAT
367
Where does pre-rRNA synthesis occur?
In the nucleolus, the nucleolus is formed when the transcription of pre-rRNA starts
368
What are the rRNA complexes that are gonna be made ex 5S
18 s 5.8s and lastly 28s
369
Nucleolus, Nucleoplasm, Cytoplasm
370
Where is the 5S from RNA Pol III transcribed?
In the nucleoplasm
371
Poly A site versus Poly A signal (AAUAAA)
Poly A signal is the AAUAAA but the actual site is the cut that's made about 20 nucleotides afterwards
372
True or false: Is 3' cleavage and polyadenylation co-transcriptional?
It is true
373
What is the splice donor sequence
GU
374
What is the splice acceptor sequence
AG
375
When splicing happens, does the snRNAs interact first or the snRNPs?
The snRNAs act first - U1 and U2 are the first to bind
376
What kind of reactions occur via the spliceosome?
Transesterification reactions
377
What is the name of the protein that binds with U2AF and binds to the branch point?
SF1
378
Which proteins will bind to the splicing site to help with specificity? What site does it bind to?
ESE DNA element which will be bound to the SR protein. The SR proteins essentially tell the members of the spliceosome that splicing is needed here
379
There are two RNA surveillance mechanisms in the nucleus what are they?
- Improperly processed mRNA will be degraded
- mRNA that still has snRNPs bound to it will not be allowed to leave
380
Nonsense-mediated decay
Normally, the stop codon appears after the final exon-exon junction.
If a premature stop codon appears before the last exon-exon junction, the cell detects this as an error (often from faulty splicing).
Exon Junction Complexes (EJCs) are left behind after splicing at each junction.
During the first round of translation, if the ribosome hits a stop codon too early and there are still EJCs downstream, NMD is triggered.
The faulty mRNA is then degraded to prevent making a truncated, potentially harmful protein.
381
What are the names of the different nucleoporins?
- Structural nucleoporins
- FG nucleoporins
- Membrane nucleoporins
382
Are the FG nucleoporins hydrophilic or hydrophobic?
Hydrophobic
383
What is the name of the nuclear import receptor
Importin
384
Is the signal sequence hydrophobic or hydrophilic? Are there positive charges nearby?
The signal sequence is hydrophobic and there are positive charges nearby
6-12 AA long
385
What is one benefit of a GPI anchor
It can diffuse freely through the bilayer. Remember, its always on the exoplasmic face
386
Which organelle generates phospholipids? Is there an issue with how it generates them?
The ER. Yes, the issue is that it can only generate phospholipids on its cytosolic face, not exoplasmic. Therefore, you need the help of scramblases (no ATP required) and flippasses to move them
387
What does PDI do?
It adds disulfide bonds to proteins. This can only be done in the ER, cannot be done in the cytosol because it is a reducing agent. It also helps add the disulfide bond in the most efficient place since sometimes they'll be added to proteins in ways that aren't energetically efficient
388
N-linked glycosylation
- Asn
- Added in ER and Golgi
- Branched structure
- Commonly added to proteins
389
O-linked glycosylation
- Ser/Thr
- Added in the Golgi
- Linear structure
- On proteoglycans
390
What protein removes the fusion of the v and t snares
NSF
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