Term
Which of the following is NOT a possible state of actin?
a. ADP-G-Actin
b. AMP-F-Actin
c. ADP-F-Actin
d. ATP-F-Actin |
|
Definition
|
|
Term
TRUE or FALSE: ATP hydrolysis is neccessary for actin polymerization to occur.
a. True
b. False |
|
Definition
|
|
Term
What occurs when the cell has a concentration of actin above Cc(+)but below Cc(-)? [Cc means critical concentration]
a. Growth occurs only from the (+) end.
b. Growth occurs at both ends.
c. No growth occurs.
d. Growth occurs at the (-) end, but not at the (+) end. |
|
Definition
| a. Growth occurs only from the (+) end. |
|
|
Term
All of the following statements about actin assembly are correct EXCEPT:
a. ADP-actin can assemble into filaments.
b. Actin subunits can treadmill through an actin filament.
c. Actin assembly can produce force for movement.
d. Actin (-) ends assemble more rapidly than actin (+) ends. |
|
Definition
| d. Actin (-) ends assemble more rapidly than actin (+) ends. |
|
|
Term
Profilin functions to:
a. Activate muscle contraction
b. Sequester actin monomers
c. Stimulate actin filament assembly
d. Cap actin filaments |
|
Definition
| c. Stimulate actin filament assembly |
|
|
Term
For a given population of microtubules undergoing dynamic instability:
a. All the microtubules will be growing at the same time
b. All the microtubules will be shortening at the same time
c. All of the microtubules will be growing at one end and shortening at the other end
d. Some of the microtubules will be growing and some will be shortening at any given time |
|
Definition
| d. Some of the microtubules will be growing and some will be shortening at any given time |
|
|
Term
Growing microtubule ends are normally "stabilized" by:
a. A GDP cap
b. A GTP cap
c. Phosphorylation of tubulin subunits
d. Gamma-tubulin |
|
Definition
|
|
Term
In cells, microtubule dynamic instability typically occurs:
a. At (+) ends only
b. At (-) ends only
c. At both (+) and (-) ends
d. At MTOCs |
|
Definition
|
|
Term
Primary culture is a good source of cells for cell biology experiments
because
a) They are mostly genetically identical
b)The connections between cells allow growth factor signals to travel
from cell-to-cell
c)They are a virtually inexhaustible source of cells
d)They respond to signals as they would as part of a larger tissue. |
|
Definition
Answers: a and d
The connections between cells are destroyed by the addition of trypsin
and calcium chelators like EDTA.
Primary culture is not an endless supply of cells. Only immortalized
cell lines provide a long-term population of clonal cells. |
|
|
Term
Inhibition of thymosin beta4 in platelet cells causes rapid actin polymerization resulting in changes in cell shape. Which of the following would also result in increased actin polymerization?
a)addition of non-hydrolyzable ATP analog (AMP PNP)
b)expression of actin subunits above the critical concentration for the minus end
c)overexpression of Arp2/3 proteins
d)inhibition of profilin |
|
Definition
The best answer here is the addition of the non-hydrolyzable ATP analog. In the presence of AMP-PNP, actin monomers would constantly be primed for addition to the filament ends. This would be similar to a situation in which profilin was very active.
This is the best answer because, as is the case with thymosinb4 inhibition, it would result in addition of subunits to existing filaments
Another good answer is c. Overexpression of Arp2/3 would result in the formation of more new filaments.
Expression of actin subunits above the critical concentration occurs in normal cells, so this condition has already been fulfilled in these cells.
Inhibition of profilin would slow filament growth because it would slow the nucleotide exchange in G-actin. |
|
|
Term
The limit of resolution for microscopy depends on the wavelength of light used. To optimize resolution, one should use:
a) UV light (short wavelengths)
b) visible light (medium wavelengths)
c) infrared light (long wavelengths) |
|
Definition
| Answer: a. Shorter wavelengths would allow a smaller limit of resolution, meaning that two objects very close together would appear distinct. At longer wavelengths, those two close objects would appear to be one object. It was also pointed out to me that we cannot see UV light, so this experiment would also require a UV light detector. |
|
|
Term
Which of the following would you expect to co-immunoprecipitate with
both actin monomers and beta-tubulin monomers?
a) alpha tubulin
b) thymosin beta4
c) phalloidin
d) alpha actinin
e) myosin II
f) none of the above |
|
Definition
Answer: f. None of the listed proteins (nor phalloidin which is not a
protein) binds to both actin and tubulin. |
|
|
Term
| How can we measure actin polymerization in vitro? |
|
Definition
Actin polymerization increases the viscosity and turbidity of the
sample. Polymerization also increases the amount of precipitate which
can also be measured. |
|
|
Term
| What are the three domains of life? What is their differences? |
|
Definition
Bacteria, Archaea, Eukaryotes.
Archaea are similar to bacteria in their energy metabolism, but are similar to eukaryotes in transcription and translation. It seems that Archaea are closer relatives to Eukaryotes than they are Bacteria. |
|
|
Term
| How can we locate our protein of interest? |
|
Definition
1. Use differential centrifugation to separate components of the cell (assuming that E and S are located in the same subcellular compartment). Cell fractionation breaks selectively the compartments of the cell and centrifuge until you are left with POI. Perform an SDS-PAGE analysis and separate POI by size (not charge).
Perform a western blot and utilize primary and secondary antibodies to locate POI.
2. Microscope approach: use microscopic techniques (eg. brightfield vs phase contrast), use dyes (usually kills cells), use fluorescent antibodies after fixing and permeabilizing the cell to visualize, use live cell imaging and molecular genetics to stick YFP or GFP etc to your POI, use electron microscopy.
3. Genetics: remove the component from the genome and see what processes are affected. |
|
|
Term
| How can we use antibodies to visualize our POI? |
|
Definition
| Isolate our POI from the cell, and inject it into a bunny. The bunny will make anyibodies specific to our POI. We can extract this antibody and then inject it into a rat. The rat will produce antibodies specific to the rabbit antibodies and is usually either radioactively labeled or has some enzyme activity that will produce light. Once we have accomplished this, we can run an SDS-PAGE with our POI, then perform a western blot utilizing the primary rabbit antibody and then afterwards, our mouse antibody. Using either radioactive sensitive film or using the enzyme activity, we can visualize the bands which contain our POI. |
|
|
Term
| Why are secondary antibodies important for identifying our POI? |
|
Definition
| Secondary antibodies provide an amplification step. Often times multiple antibodies will bind to a single protein. Afterwards, many secondary antibodies can bind to a single primary antibody. This amplifies the signal and reduces the noise to signal ratio. |
|
|
Term
| How is a western blot performed? |
|
Definition
| We first run an SDS-PAGE with our POI. Then we use a nitrocellulose film to bind the protein on the SDS-PAGE gel. This provides a negative "image" of the SDS-PAGE gel. The next step is to wash the film with a dilute protein solution in order to have proteins bound evenly to the nitrocellulose. If we didn't do this and we washed the film with our antibody, the antibody would stick everywhere there were no proteins bound in addition to our POI. After blocking the film, we add our primary antibody followed by our secondary antibody. Add either a substrate for the enzyme on the secondary antibody or simply use radioactive sensitive film to capture the bands on the film with our POI. |
|
|
Term
| What are the differences between brightfield, phase contrast, nomarsky and darkfield microscopy? |
|
Definition
Brightfield: full spectrum of light passes through the sample. Usually requires a dye for any appreciable resolution.
Phase contrast: Rings bend the light so that we get more contrast on the sample. This is useful for looking at unstained samples.
Nomarski optics (DIC): light is polarized and allowes us to see more topography. It exaggerates differences in the cell.
Darkfield: Light is directed in at the sample from the side and is scattered by the sample. This is also useful for unstained samples. |
|
|
Term
|
Definition
| Resolution has to do with whether 2 objects are distinct or not. i.e. how close 2 objects can be and still appear to be separate. |
|
|
Term
| What is the limit of resolution? |
|
Definition
| D= (0.61)lambda/(n*sin(theta)); lambda is the wavelength of light, n= refractive index btw sample and lens, theta= 1/2 angular width. A small number D indicates a good limit of resolution. |
|
|
Term
| What steps must be taken in order to visualize an organelle in a cell? |
|
Definition
1. Fix cell (cross link proteins)
2. Permeabilize the cell membrane
3. Add primary antibody
4. Add secondary antibody
5. Add substrate if an enzyme is attached to the secondary antibody. |
|
|
Term
| How can we figure out if 2 proteins co-localize within the cell? |
|
Definition
| Use one antibody with one color to tag one protein, and use a different antibody with a different color to tag our other POI. Check if the colors co-localize. 2 proteins that associate together probably function together. |
|
|
Term
| What happens to the wavelength of light that is used for fluorescence microscopy? |
|
Definition
| A filter is used to only allow 480nm light through the sample. If any GFP is present, it will absorb the wavelength of light and emit 510nm light. Another filter must be used to filter out all light besides ones at the wavelength of 510nm. Multiple filters can be used to take multiple pictures to see where each protein resides. |
|
|
Term
| Name the fluorescent dyes: |
|
Definition
DAPI: binds DNA, emits 460nm.
Rhodamine: Doesn't bind intrinsically, absorbs 540nm and emits 620nm.
Fluoescien: absorbs 500nm, emits 520nm. |
|
|
Term
| How is the noise to image ratio improved in fluorescent microscopy? |
|
Definition
Detectors can amplify structural features and reduce background noise.
1. Confocal images: thin sections of samples or thin sections of light are used.
2. Deconvolution: digitally remove noise and amplify signal |
|
|
Term
| How can we visualize live cells? |
|
Definition
1. Differential image/ Nomarsky optics can be used to see differences in refractive indices of the cell.
2. Use GFP, etc. to insert into the end of a gene encoding for your POI to see it. |
|
|
Term
| How can we use genetics to find our POI? |
|
Definition
| Use molecular genetics to fuse gene of interest with plasmid to insert gene for GFP. We usually use electroporation (induces pores in PM) to get the plasmid into CHO (Chinese hamster ovary cells). This transforms the cells. Problems associated with this technique include that GFP is big and has its own characteristics. Sometimes it hinders proper movement of the protein through membrane transporters. Also, the promoter can either yield too little GFP or too much GFP to be distinguished from background noise. Need to use multiple techniques to verify the results. |
|
|
Term
| How does electron microscopy work? |
|
Definition
| The sample is treated, fixed (chemically or with liquid Helium). This allows the water in the cell to become vitrous ice (ice that is not crystalized and distorted) which creates life-like preservation. The cell is then permeabilized or saturated with heavy metals (gold, silver, lead, uranium). Then make a thin section of the sample with an ultra microtome. Stick it into a vacuum and have your antibody bind to the heavy metal. The electron beam is steared with electromagnetic lenses and condensers. This is very expensive and not practical for most applications. |
|
|
Term
| How can we tell if two separate proteins bind to one another? |
|
Definition
Co-immunoprecipitation:
(like affinity chromatography) Beads coated with antibodies will bind selectively to one of the two proteins you are studying. If both proteins are found in the pellet after centrifugation, we know that they bind to one another. We can test this by performing SDS-PAGE runs and comparing bands to where they should be and where they actually are. Western blot confirms results. |
|
|
Term
| How can we test for co-localization in the cell? How can we test whether 2 proteins are in the same complex? |
|
Definition
Use fluorescent microscopy to check for overlap of signal. If there is no overlap, but they can bind to each other it shows that they can interact, but not in a live situation.
For molecules in the same complex, we must use FRET. |
|
|
Term
| What is FRET and how does it work? |
|
Definition
| FRET= Fluorescence Resonance Energy Transfer. What happens is, we can pick a wavelength of light such as 450nm that is absorbed by CFP. CFP then in turn emits a wavelength of 480nm. If the molecule of CFP happens to be within 0.5nm away from a molecule of YFP, then the molecule of YFP will absorb the light and emit 530nm light. Therefore, if you detect 530nm light while shining 450nm light on a sample, you know that your proteins are located within the same complex. |
|
|
Term
| How can we tell if the localization of protein (A) depend on the localization of protein (B)? |
|
Definition
1. Genetics: Delete protein (B) from the genome.
2. RNAi: Use complementary RNA transcripts to base pair with the mRNA encoding for protein (B) and targets it for degradation.
3. Cell fractionation and co-immunoprecipitation.
4. Co-localization by immunofluorescence
5. Genetics: destroy prenylation site (site for lipid linker attachment) which is usually a cysteine residue on the C-terminus.
NOTE: Most of these techniques are best used with one another in order to be certain of different results. |
|
|
Term
| What are the 3 types of cellular filaments? |
|
Definition
1. Actin
2. Intermediate Filaments
3. Microtubules |
|
|
Term
| Where are each of the 3 filaments located in the cell? |
|
Definition
Actin: coats the cortex of the cell (just inside the PM)
Intermediate filaments: Coat inside of the Nuclear Membrane and facilitate cell-cell contact.
Microtubules have a vertical arrangement that creates polarity which determines the top from the bottom of the cell. Microtubules also construct the mitotic spindle and the contractile ring for cytokinesis. |
|
|
Term
| What are the functions of Actin? |
|
Definition
| Actin is used in muscle contraction, cell crawling, cytokineses, cell shape, etc... |
|
|
Term
|
Definition
| Actin is made up of assymetric monomers of actin. Actin monomers can bind ATP and hydrolyse it to ADP. The actin filament is made of 2 parallel strings of actin that come together. |
|
|
Term
| How can we determine how much G-Actin is in a tube versus F-Actin? |
|
Definition
G-Actin is globular actin and F-Actin is actin which is bound in a filament. We can determine how many filaments are formed by three tests:
1. Turbidity- How clear the solution is
2. Viscosity- More viscous=more filaments
3. Precipitation: size of pellet from centrifugation. |
|
|
Term
| What is the critical concentration of actin mean? |
|
Definition
| There is a minimum concentration of G-Actin that must be present in a sample in order for a nucleation site to form for actin polymerization to occur. |
|
|
Term
| What is more likely to polymerize into an actin filament: ADP bound actin monomers or ATP bound actin monomers? |
|
Definition
| ATP bound monomers have a much higher affinity for a growing filament than ADP bound monomers. As the filament ages, the ATP is hydrolyzed to ADP and this acts as a timer for the cell. (allows the cell to know how old an actin filament is) Actin filaments usually have an ATP cap at the ends. |
|
|
Term
| Which end do actin monomers add more rapidly? |
|
Definition
| They add faster to the + end and slower to the - end. |
|
|
Term
|
Definition
| Actin monomer concentration is above the critical concentration for + end polymerization, but below the critical concentration for the - end. This results in a net gain from the + end and a net loss from the - end, causing a treadmilling effect. |
|
|
Term
| In what 7 ways is actin polymerization regulated by proteins? |
|
Definition
1. Bind actin monomers to nucleate actin filaments.
2. Bind G-Actin monomers and control ATP/ADP state (nucleotide exchange factor).
3. Bind monomers and sequester them so that they cannot form filaments.
4. Bind to the end of the filament to inhibit the growth of the filament.
5. Bind to the side of the filament to inhibit growth.
6. Proteins in which bind to the side of a filament and cut them.
7. Proteins that bind to sides of filaments and cross-link them together (very specific). |
|
|
Term
| What are the actin nucleators and how do they work? |
|
Definition
1. Arp 2/3: an actin related protein. It creates a favorable pseudo - end that allows G-Actin to bind.
2. Formin: Associates with the + end of the filament and has a high affinity for actin monomers bound with ATP. Formin gets the monomer close and oriented to the + strand so that subunit addition is favorable. |
|
|
Term
| What complex binds G-Actin to control its ADP/ATP state and how does it work? |
|
Definition
| Profilin: It binds to G-Actin, induces ADP release, and thereby allows a new molecule of ATP to bind. It works together with formin. |
|
|
Term
| What binds and sequesters Actin monomers? |
|
Definition
| Thymosin: It binds G-Actin and blocks any interaction it can have with the filament. This is higly involved in blood clotting. Clotting factors inhibit thymosin function and make massive changes in filament shape to induce clotting. |
|
|
Term
| What is Arp 2/3 and what does it do? |
|
Definition
| Arp 2/3 is an actin nucleator. It is an actin related protein that provides a nucleation site for actin polymerization to occur. |
|
|
Term
| What is formin and what does it do? |
|
Definition
| Formin is a complex that associates with the + end of an Actin filament. There it has a high affinity for actin monomers bound with ATP and facilitates subunit addition. |
|
|
Term
| What is profilin and what does it do? |
|
Definition
| Profilin is a nucleotide exchange factor that facilitates the exchange from ADP bound G-Actin to ATP bound G-Actin. It usually works together with formin to have a large imact on assembly. |
|
|
Term
| What is thymosin and what does it do? |
|
Definition
| Thymosin binds G-Actin and blocks its interaction with the growing actin filament. It is highly involved in blood clotting. |
|
|
Term
| What functions to sever actin filaments? |
|
Definition
Cofilin: It binds to the side of the filament and causes a twist. This twist destabilizes its interactions between subunits. It preferentially binds to ADP bound subunits.
Gelsolin: Binds to the middle and cuts filaments. It then stays associated with the + end of that piece. It makes things more "soluble". |
|
|
Term
| What is cofilin and what does it do? |
|
Definition
| Cofilin severs actin filaments. It preferentially chooses ADP bound subunits, creates a twist in the actin strand and destabilizes it causing it to break. |
|
|
Term
| What is gelsolin and what does it do? |
|
Definition
| Gelsolin binds to the middle of actin strands and cuts them. It stays associated with the + end of that filament. |
|
|
Term
| What complexes bind filament ends? |
|
Definition
1. + end capping proteins:
-Cap z: binds to the + end and limits subunit addition and subunit loss at the + end.
2. - end binders:
-Tropomodulin: binds to - end and limits subunit addition and loss from - end. |
|
|
Term
| What is cap z and what does it do? |
|
Definition
| Cap z is a + end capping protein that limits subunit addition and loss at the + end. |
|
|
Term
| What is tropomodulin and what does it do? |
|
Definition
| Tropomodulin is a - end capping protein that limits subunit addition and loss at the - end. |
|
|
Term
| What complex binds to the side of actin and stabilizes it? |
|
Definition
| Tropomyosin: It binds 7 actin subunits and stabilizes it. If it is present then obviously cofilin can't bind. |
|
|
Term
| What is tropomyosin and what does it do? |
|
Definition
| It binds to the sides of actin filaments and stabilizes 7 actin subunits. It acts to stabilize the filament. |
|
|
Term
| What proteins act to bind and crosslink actin chains? |
|
Definition
1. alpha actinin: makes loose bundles of parallel actin.
2. fimbrin and villin: make tight bundles of parallel actin.
3. Filamin: Dimer that arranges actin filaments into a gel-like network. This dimer has one side where it binds to itself and the other side binds to the actin molecule. This helps with the spacing and angle of actin. |
|
|
Term
| What is alpha actinin and what does it do? |
|
Definition
| Alpha actinin is a protein that binds and cross links chains of actin. It creates parallel loose bundles. |
|
|
Term
| What is fimbrin and what does it do? |
|
Definition
| Fimbrin works together with villin to bind and crosslink actin. It acts to create tight parallel bundles of actin. |
|
|
Term
| What is villin and what does it do? |
|
Definition
| Villin works together with fimbrin to bind and crosslink actin. It acts to create tight parallel bundles of actin. |
|
|
Term
| Name the drugs associated with actin: |
|
Definition
1. Phalloden: drug that binds between actin subunits and strengthens their interaction. We can take phalloden and attach rhodamine so that we can visualize actin with light microscopy. For comparison, if we attached rhodamine to an actin antibody, all actin monomers would be visualized as well as actin strands. This would be messy.
2. Latrunculin A binds G-actin monomers and prevents them from adding to actin filaments. Long term this destroys actin networks.
3. Cytochalasin D binds to the plus ends of actin filaments, caps them, and prevents further polymerization. This drug also destabilizes existing filaments. |
|
|
Term
| What is cytochalasin D and what does it do? |
|
Definition
| Cytochalasin D is a drug that binds to the plus ends of actin filaments, caps them, and prevents further polymerization. This drug also destabilizes existing filaments. |
|
|
Term
| What is latrunculin A and what does it do? |
|
Definition
| Latrunculin A is a drug that binds G-actin monomers and prevents them from adding to actin filaments. |
|
|
Term
| What is phalloidin and what does it do? |
|
Definition
| Phalloidin is a drug that binds to actin filaments and stabilizes them. Since actin monomers can still add to the ends of these filaments, phalloidin treatment results in net actin assembly. |
|
|
Term
| Name the actin based and microtubule based motor proteins: |
|
Definition
Actin based: myosins
Microtubule based: kinesin and dynein |
|
|
Term
| What is myosin II and what does it do? |
|
Definition
Myosin II is a motor complex with 2 domains. It has 2 motor heads made up of the heavy chain and 2 tails that associate with one another that constitute the light chains.
It is a + end directed motor protein that uses ATP hydrolysis to move. |
|
|
Term
| What is the basic contractile unit? |
|
Definition
| The sarcomere. Many sarcomeres come together to make a myofibril. |
|
|
Term
| What is the thin filament, the thick filament and the z disc? |
|
Definition
| The thin filaments are actin, the thick are myosins and the z disc is made of alpha actinin. |
|
|
Term
| Myosin moves towards which end on actin? |
|
Definition
|
|
Term
| Name the important filaments and compounds present in a sarcomere: |
|
Definition
z-disc
tropomodulin: caps the - ends of actin
cap z: caps the + ends of actin
alpha actinin: cross links actin
titin: keeps thick filament in the center
actin
nebulin: binds actin and helps determine its length
thick filament: myosins |
|
|
Term
| Name the processes which happens with respect to a myosin head during a muscle contraction: |
|
Definition
1. Myosin head is attached to actin with ADP bound.
2. ADP dissociates
3. ATP binds and myosin head is released
4. ATP is hydrolyzed and myosin head becomes cocked
5. Myosin head attaches to the actin filament
6. Inorganic phosphate releases causing tight binding and the myosin head to perform the power stroke.
7. The cycle repeats |
|
|
Term
| What is tropomyosin and what does it do? |
|
Definition
| Tropomyosin associates with actin and blocks myosin binding. |
|
|
Term
| What functions to block myosin binding? |
|
Definition
|
|
Term
| What is troponin and what does it do? |
|
Definition
| Troponin is a CTI trimer that binds actin, tropomyosin and calcium. When calcium ions find troponin, it causes a conformational change and moves tropomyosin away from the myosin binding sites on actin. |
|
|
Term
| What is myosin I and what does it do? |
|
Definition
| Myosin I is a single headed motor protein that binds actin and membranes. It functions to move membranes for growth or for cilia movement. |
|
|
Term
| What is myosin II and what does it do? |
|
Definition
| Myosin II is a 2 headed motor protein that is responsible for muscle contraction and cytokinesis. |
|
|
Term
| What is myosin V and what does it do? |
|
Definition
| Myosin V is a motor protein that is responsible for organelle movement and subcellular organization. |
|
|
Term
| What is myosin VI and what does it do? |
|
Definition
| Myosin VI is a motor protein that is rare and is responsible for some - end directed actin movement. |
|
|
Term
| Are alpha and beta tubulin linked, covalently bonded or simply associate? |
|
Definition
|
|
Term
| On which part of the tubulin dimer is GTP hydrolyzable? |
|
Definition
| The beta subunit has catalytic activity but the alpha subunit does not. |
|
|
Term
| How many protofilaments make up a microtubule? |
|
Definition
|
|
Term
| What adds faster: GTP bound tubulin or GDP bound tubulin? |
|
Definition
| GTP bound tubulin because GTP bound tubulin has a conformational difference in which addition is unfavorable. |
|
|
Term
| What does dynamic instability mean? |
|
Definition
| For a given population of microtubules, the majority can have a net polymerization, but a single microtubule can have an array of catastrophes and rescues. In vitro, most catastrophes result in complete filament loss. |
|
|
Term
| What are gamma TuRCs and what do they do? |
|
Definition
| Gamma TuRCs are rings of gamma tubulin that look like a spring washer and are held together by accessory proteins. Tubulin dimers use this as a scaffold to nucleate and form microtubules. The - end of the microtubule is where the gamma tubulin is located. Usually gamma TuRCs are located around centrosomes (MTOCs) |
|
|
Term
| What are centrioles and what do they do? |
|
Definition
| Centrioles are two perpendicular cylinders of 9 microtubule triplets. They exist at the MTOC. |
|
|
Term
| What bind and nucleate microtubules? |
|
Definition
|
|
Term
| What are proteins that bind tubulin dimers and sequester them to prevent microtubule assembly? |
|
Definition
| Staffamen: binds 2 dimers at a time. |
|
|
Term
| What is staffamen and what does it do? |
|
Definition
| Staffamen binds tubulin dimers and sequesters them to prevent microtubule assembly. |
|
|
Term
| What proteins bind and sever existing microtubules? |
|
Definition
| Katanin: Dimer that uses one subunit to ATP to cut the microtubule, the other binds to the MTOC. This is good for dissasembly of microtubules during cell division. |
|
|
Term
| What is katanin and what does it do? |
|
Definition
| Katanin is a dimer that cuts microtubules. One subunit uses ATP hydrolysis to cut the microtubule while the other subunit binds to the MTOC. It is useful to take down the microtubule network for cell division. |
|
|
Term
| What are the microtubule associated proteins (MAPs)? |
|
Definition
1. Tau: binds to multiple microtubules and bundles them together.
2. MAP2: binds to make loose bundles.
3. Misc. bind to stabilize microtubules. |
|
|
Term
| What are the microtubule associated proteins (MAPs)? |
|
Definition
1. Tau: binds to multiple microtubules and bundles them together.
2. MAP2: binds to make loose bundles.
3. Misc. bind to stabilize microtubules. |
|
|
Term
| What is tau and what does it do? |
|
Definition
| Tau binds multiple microtubules together to form tight bundles. |
|
|
Term
| What proteins bind microtubule ends? |
|
Definition
1. XMAP 215: binds ends and stabilizes them to prevent loss of subunits.
2. Kinesan B: binds microtubule ends and promotes subunit loss.
3. +TIPS: plus end tubulin proteins that interact with microtubules and membranes. |
|
|
Term
| What are the drugs that regulate microtubules? |
|
Definition
1. Nocadozole: binds subunits and prevents assembly.
2. Colchicine: binds subunits and prevents assembly.
3. Toxol: binds and stabilizes microtubules. This can stop cell division because the cell can't get rid of it's microtubule network. |
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Term
| What is kinesin and what does it do? |
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Definition
| Kinesin is a + end directed motor protein that is related to myosin. The major difference between the two lies with a light chain on one end of the coil-coil interaction that can bind to cargo for transport. It specifically binds to beta tubulin and not alpha tubulin. |
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Term
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Definition
ATP hydrolysis is utilized:
1. ADP bound front head binds beta tubulin and releases ADP (tighter binding).
2. ATP binds and causes the back head to be thrown forward.
3. ATP in now rear head is hydrolyzed and inorganic phosphate is released.
4. ADP bound front head binds to beta tubulin and the process is repeated. This causes it to be highly processive since it is constantly bound to the microtubule unlike myosin on actin. |
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Term
| What is kinesin 5 and what does it do? |
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Definition
| Kinesin 5 is a tetramer with 4 heavy chains that help anaphase B by pushing opposing MTOCs towards opposite ends of the cell. + end directed. |
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Term
| What can genetic recombination tell us about conventional kinesins? |
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Definition
1. N-terminal motors create + end directed motor proteins.
2. C-terminal motors create - end motor proteins.
3. Central motor coding do not walk, instead they destabilize microtubule filaments. |
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Term
| What is dynein and what does it do? |
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Definition
| Dynein is a - end directed motor protein with no homology to that of kinesin. It is a huge protein complex that is not as processive, but takes more steps than kinesin. There are 2 types: Cytoplasmic dynein and axonemal dynein (not axons, actually microtubule based protrusions of the cell). |
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Term
| What cargo does dynein carry? |
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Definition
| Dynein carries vessicles retrograde and also plays a role in golgi localization. |
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Term
| What is dynactin and what does it do? |
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Definition
| Dynactin is a dynein activator that is homologous to Arp 1. It determines what cargo binds and when dynein moves. |
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Term
| What are axonemes and what do they do? |
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Definition
| Axonemes are not axons. They are cell protrusions that allow movement in many cell types and sometimes provide sensation. |
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Term
| What is the structure of a flagellum? |
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Definition
| It is a 9+2 microtubule arrangement that has inner dyneins with 2 heads and outer dyneins with 3 heads. It also has a compound called nexin that binds microtubule doublets together. |
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Term
| What is nexin and what does it do? |
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Definition
| Nexin is a protein that binds microtubule doublets together in the flagella. Without it, the filaments would slide past each other, making it impossible to swim. |
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Term
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Definition
| Axonemal dependent movement. It uses microtubule based structures and motors. |
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Term
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Definition
| Crawling is a actin dependent cell movement. |
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Term
| How does cell crawling occur? |
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Definition
Actin from the cell cortex forms stress fibers which attach the cell with integrins to the substrate.
1. Protrusion: actin polymerizes to move front edge of the cell forward.
2. Attachment: the actin sets up new stress fibers and substrate contacts.
3. Traction: The cell removes the previous stress fibers and moves forward. |
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Term
| What are intermediate filaments responsible for? |
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Definition
1. Cell adhesion (connect to one another and substrate)
2. Nuclear lamins (protect nucleus from mechanical damage)
3. Cell constructs (keratins cross link to form stable structures like hair, fingernails, etc...) |
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Term
| What does phosphorylation do to nuclear lamins? |
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Definition
| It dissasembles them from their network. |
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Term
| Where are nuclear lamins found? |
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Definition
| All cells from a species with a vertebrae. |
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Term
| Where are keratins found? |
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Definition
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Term
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Definition
| Muscle cells for cell/cell connections. |
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Term
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Definition
| Fibroblasts (dynamic during cell division) |
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Term
| Where are neuro-filaments found? |
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Definition
| In neurons (these are intermediate filaments) |
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Term
| What is the structure of intermediate filaments? |
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Definition
| It is composed of 2 parallel dimers in an antiparallel fashon. Therefore it is a non-polar tetramer made up of 2 acidic alpha helices and 2 basic alpha helices. 8 protofilaments make up an intermediate filament. |
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Term
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Definition
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Term
| How does the amount of neurofilaments correlate with the size of the axon? |
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Definition
| The more neurofilaments there are, the larger the diameter of the axon becomes. |
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Term
| What is plectin and what does it do? |
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Definition
| Plectin links intermediate filaments to microtubules and actin. Mice deficient in plectin have many many many problems (usually don't survive). |
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Term
| How do intermediate filaments associate with one another? |
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Definition
| The c-terminal domain sticks out on NFL-NFM pairs and forms a filament-filament interaction. |
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Term
| How is cell crawling different from cell swimming? |
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Definition
| Cell crawling occurs with actin polymerizing, whereas swimming results in motor function on microtubules. |
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Term
| Name the 3 types of cell protrusions: |
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Definition
1. Filopodia: small protrusions that contain tightly bundled, parallel actin filaments.
2. Lamellopoeida: wide, flat sheet that contains branched actin networks.
3. Pseudopodia: stubby 3 dimensional feet with gels of actin.
The common difference is in the type of actin polymerization that occurs. |
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Term
| What are keratocytes and what do they do? |
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Definition
| Keratocytes are mobile cell types. |
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Term
| Where are each found in a lamellopoida of a keratocyte: Arp 2/3, Cofilin, actin and myosin II? |
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Definition
1. Arp 2/3 is located at the leading edge. Here it mitigates actin growth and polymerization in branched 70 degree networks.
2. Cofilin binds ADP bound subunits and breaks the filaments. This is located towards the cell body at the inside edge of the lamellopoida.
3. Myosin II is an actin based motor that is located at the opposite end of the cell body and functions to propell the cell forward by "pushing the toothpaste out of the tube". |
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Term
| What is rho and what does it do? |
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Definition
| Rho is part of a family that are small, inefficient GTPases. They act as molecular switches, actin on when bound to GTP and off when bound to GDP. GAP (GTPase activating protein) acts to switch GTP-Rho to GDP-Rho and opposite, GEF (GTP exchange factor) acts to make GDP-Rho into GTP-Rho. |
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Term
| What happens when we insert a dominantly active Rho protein into a cell? |
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Definition
| Rho acts continually active because it neither hydrolyses ATP, nor interacts with GAP. Because of this the cytoskeleton becomes saturated with stress fibers. |
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Term
| What happens when we insert a dominantly active Rac protein into a cell? |
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Definition
| The dominantly active version creates a large lamellopodium with high concentrations of actin at the leading edge. |
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Term
| What does Rho favor? Rac? |
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Definition
Rho: stress fibers
Rac: lamellapodia |
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Term
| What does Rho favor? Rac? |
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Definition
Rho: stress fibers
Rac: lamellapodia |
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Term
| What does Rho favor? Rac? |
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Definition
Rho: stress fibers
Rac: lamellapodia |
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