Lectures 1-7 Flashcards

Question 1

Q

Why do we study cells?

Answer

A

to determine the normal so that we can study the abnormal
the cell is the fundamental unit of life
to understand how the systems and organelles within a cell work and cooperate to enable cells to function autonomously and in tissues

Question 2

Q

What is Cell Culture?

Answer

A

Cell Culture is a technique used to grow cells or tissues outside their organism under strictly controlled conditions.

Step 1: Clump –> individual cells (isolate by breaking cell-cell and cell-matrix interactions using EDTA, mechanical fragmentation, and trypsin.
Step 2: Grow cells in a medium+serum (filled with nutrients, insulin, growth factors, etc.) in a 37C CO2 incubator.
- cells can grow as Adherent Culture or Suspension Culture

Question 3

Q

What is EDTA?

Answer

A

A chelating agent that removes metal ions, used for isolating cells.

Question 4

Q

What is Primary Cell Culture referring to?
What is the Hayflick Limit? Why does it happen?
If cell density is high what happens?

Answer

A

when cells are taken directly from an organism – can usually only divide around 50 times/generations (“Hayflick limit”) because of the shortening of telomeres
If cell density high: contact inhibition – they’re so pressed up together that they stop dividing

Question 5

Q

What’s the opposite (why do I say this?) basically of Primary Cell Culture?

Answer

A

Cell line because they are able to divide indefinitely (aka, immortal/cancer cells)

less likely to exhibit contact inhibition + they have telomerase so they don’t need to stop dividing
HeLa was the first human cell line

Question 6

Q

What would a Transformed culture look like?

Answer

A

hair-like processes
rounded instead of elongated
disorganized, they grow on top of each other instead of in parallel arrays
loss of contact inhibition
turn media acidic because they deplete the nutrients from growing so fast — only way growth stops

Question 7

Q

What’s the difference between a stem cell and a progenitor cell?

Answer

A

Stem cells can give rise to more than one type of cells, progenitors cannot.

Question 8

Q

What’s a differentiated cell?

Answer

A

The end of the line result. It cannot divide anymore to give rise to another cell.
Transcription Factors decline dramatically in differentiated cells.

Question 9

Q

Compare Asymmetric Cell Divisions vs Symmetric Cell Division

Answer

A

Symmetric: gives rise to 2 identical cells to mom
Asymmetric: 1. one is similar, one is different. 2. both are different

Question 10

Q

Embryonic Stem Cells Growth Process

Answer

A

get a cultured blastocyst. Put the inner cell mass from that into a dish containing Fibroblast Feeder Cells (which are helper cells that help the ICM to survive). Once they survive and form established cultures we call them “Embryonic Stem Cells/bodies” and put them in suspension culture.

Question 11

Q

What does pluripotent mean?

Give an example

Answer

A

Can be grown indefinitely in culture in appropriate conditions. And can give rise to the 3 types of cells form the 3 germ layers.
Embryonic Stem cells are pluripotent
iPS cells are pluripotent

Question 12

Q

Adult Stem Cells in Intestinal Epithelium
Purpose?
Where are they located?
Properties?

Answer

A

Most tissues contain Adult Stem Cells. They’re required bc they maintain and repair the tissue in which they are located.
They are robust and can divide rapidly (we replace our intestinal epithelium every 5 days)
They are capable of generating a limited # of different cell types
Located in stem cell niche (in the crypt)– they’re surrounded by adjacent cells that secrete factors which tell the ASCs to either renew or differentiate .
Differentiated stem cells are lost (die?) at the tip of the vili

Question 13

Q

Explain the concept of a One-Way Trip when referring to differentiated stem cells.
What’s the new finding?
How did they discover?
Medical application of this new finding.

Answer

A

Usually when you go from Stem Cell to differentiated cell, you can’t go back.
BUT: mature differentiated normal cells can be reprogrammed to become induced pluripotent stem cells (iPS cells).
You reprogram Fibroblasts by introducing the 4 Yamanaka Factors into the the differentiated cells.
Transcription Factors decline dramatically in differentiated cells. But if we artificially put high levels of TFs in them (using viruses/viral factors), some will revert back.
These iPS cells can be made patient-specific and can be differentiated into affected neurons to be screened to be used for drugs

Question 14

Q

What are the 3 germ layers?

Answer

A

Endoderm, Mesoderm, Ectoderm

These different layers give rise to different cells/tissues

Question 15

Q

Safety Concerns with Yamanaka Factors

Answer

A

The use of viral vectors is risky because it could potentially lead to the virus integrating into new cells, and then the factors make the new cells transform and divide uncontrollably.

Question 16

Q

Bright-Field Microscopy

what’s the condenser lens, objective lens, ocular/eyepiece lens for?
what thing does the specimen have to have in order to be able to see?

Answer

A

only structures with a high refractive index are observable
MAGNIFICATION 1000X
condenser lens to focus light on specimen
objective lens to collect light 100X
eyepiece/ocular lens to focus image onto eye 10X

Question 17

Q

Bright-Field Microscopy
- what’s the condenser lens, objective lens, ocular/eyepiece lens for?
What’s the limit of resolution of this microscope?

Answer

A

only structures with a high refractive index are observable
MAGNIFICATION 1000X
condenser lens to focus light on specimen
objective lens to collect light 100X
eyepiece/ocular lens to focus image onto eye 10X

200nm or 0.2um is the limit of resolution

Question 18

Q

Resolution Equation

What does each mean?

Answer

A

The smaller the D the better
D=(0.61 lambda)/(Nsin alpha)
alpha is the 1/2 angle of light entering the objective lens
N= refractive index of medium btwn specimen and objective lens (higher the better)
Lower/longer the wavelength the better

Question 19

Q

How does contrast play a part in light microscopy?

Answer

A

When waves passing through cell are in phase, the light is bright. When the light passes through the nucleus, for example, it has a high refractive index and through INTERFERENCE causes the light to be 1/4 out of phase —> dimmer light

Question 20

Q

How does contrast play a part in Phase Contrast microscopy?

Answer

A

When waves passing through cell are in phase, the light is bright. When the light passes through the nucleus, for example, it has a high refractive index and through INTERFERENCE causes the light to be 1/4 out of phase —> dimmer light
Then the phase plate also gives 1/4 out of phase === 1/2 wavelength out of phase light on image plane

You get the halo bc cells aren’t perfectly rounded, so wavelengths superimpose, creating a high amplitude, which is bright light

Question 21

Q

Differential Interference Contrast Microscopy
What gives the contrast?
What is it used for?
One characteristic that can be seen in pics

Answer

A

to examine live unstained cells. small differences in N are exaggerated to be seen as contrast by the eye.

uses polarized light (has polarizers that separate the plains of light. The interference when the plains are recombined gives the contrast.)

Question 22

Q

What is Phase Contrast used to examine.

One characteristic that can be seen in pics

Answer

A

to examine live unstained cells

HALO and dark spot=nucleus

Question 23

Q

Fluorescence Microscopy
Advantage?
How are the images obtained?

Answer

A

advantage: can image more than one type of cell structure because fluorochromes . Allows us to locate structures – by conjugating a fluorochrome to Ab (Immunofluorescent Staining).
White light source passes through excitation filter which lets out only the optimal wavelength. Then the specimen absorbs it and emits light through emission filter onto image plane.

Observes fixed/dead cells

Question 24

Q

What is a fluorochrome?

Answer

A

A dye that fluoresces. absorbs E, which kicks e into a higher unstable orbital. Then it goes back down, emitting light

Question 25

Q

What’s the Stokes Shift?

Answer

A

The difference between the excitation and emission lights

Question 26

Q

Explain Immunofluorescent Staining and Microscopy

What types of cells do you do this on?

Answer

A

Prepare a sample of your protein on microscope slide. incubate with primary Ab and wash away all unbound Abs. Incubate with fluorochrome-conjugated secondary Ab (which will make primary Ab locatable). Wash away unbound and observe in fluorescence microscope.

Observes fixed/dead cells!

Question 27

Q

Dual Fluorescence Microscopy

Answer

A

is a type of Immunofluorescence microscopy, but you use chemicals instead of Abs. You do two labelings and images and then digitally overlay them.
Use phalloidin- a fluorochrome-conjugated drug that binds actin filaments and fluoresces red.

Question 28

Q

Green Fluorescent Protein (GFP)

Answer

A

allows for fluorescent imaging in live cells
from jellyfish. Fluoresces green when shone with blue.
you can fuse GFP with your gene of interest, that way RNA Polymerase can recognize it and transcribe it into GFP-Fusion Protein
you can even mutate certain AAs within GFP to make fusion proteins with different excitation&emission profiles
- which allows us to coexpress proteins and visualize them simultaneously

Question 29

Q

Laser Scanning Confocal Microscope

Answer

A

uses confocal pinholes to remove the out-of-focus light, because only the in-focus fluorescent light from the specimen can pass through to get to detector.
uses Optical Sectioning (illuminates one point at a time and then combines the images)
good 3D resolution but time and money expensive

Question 30

Q

What’s a cheaper option that Laser Scanning Confocal Microscopy?

Answer

A

Deconvolution Microscopy uses the mathematical Point Spread Function to remove blurriness from fluorescence by robot taking picks at different focal planes (making a Z Stack). The robot compares the images to themselves and a reference set of beams to figure out what the relative focus is and what is out of focus.
Deconvulves the images

Question 31

Q

What’s Two-Photon Excitation Microscopy?

Answer

A

an alternative to Laser Scanning Confocal Microscopy because you don’t need confocal pinholes anymore. Instead you just sequentially shoot two photons of light at half the wavelength (960nm)of the light you would have done if you were using a single photon (488nm). The specimen still generates the same emission wavelength

Scans deep tissue

Question 32

Q

Fluorescence Resonance Energy Transfer (FRET)

Answer

A

to measure protein interactions in live cells. Piggy back system

If the protein attached to CFP (cyan fluorescent protein) interacts with protein attached to YFP you get yellow fluorescence when CFP is excited because FRET happens between CFP and YFP

If not, CFP gets excited and just emits cyan-coloured light

You’d set the FRET filter at what excites CFP

Question 33

Q

Electron Microscopy
What types of cells are we imaging?
Compare to Fluorescence Microscopy
How is the image formed?

How can it be detected in the image?

Answer

A

used on dead/fixed cells that are sectioned and/or metal-coated
uses electrons from a wire filament that rush towards the anode, not light
waaay better resolution than FM
uses magnetic condenser, instead of glass
dark spots in image: electrons deflect off portions coated with heavy metal bc they’re electron-rich – no penetration
bright spots in image: electrons penetrate sample, and are focused onto a phosphorescent screen that has crystals in it which are excited and thus give off light energy.

You’ll be able to see the proteins and membranes

Question 34

Q

What are the two types of Electron Microscopy?

What’s the Resolution equation for this?

Answer

A

Scanning EM
- based on the angle of deflection of the scattered electrons to give info about 3D structure
- can recognize because looks super 3D
Transmission EM
- based on what passes through the specimen (what does is registered as light, what deflects is registered as dark)
- D= (0.61 lambda)/ alpha —– waaay better resolution (because electrons have such a smaller wavelength than light)

Question 35

Q

What’s Immunoelectron Microscopy?

Answer

A

Use of Electron Microscopy to detect specific electrons in a specimen by conjugating Abs (bound to your protein) to gold particle (heavy metal), so that the electrons deflect off it, showing dark spots where the protein of interest is

Question 36

Q

How would you label live T-cells intracellularly and extracellularly?
What’s the next step?

Answer

A

Use fluorescent fluorophores and stains.

bind Abs to transmembrane or surface proteins. Then bind coloured (r or green) flurophores to those Abs
or label cells intracellularly by staining the organelles (ex, Nucleus with Hoechst Stain, which is a membrane-permeable dye)

These labelled cells can now go on to be sorted and counted, then analyzed

Question 37

Q

How would you sort cells after being labelled?
How would you count the cells?
What’s another way to use FACS?

Answer

A

Use FACS: Fluorescent Activated Cell Sorting to sort the cells by the degree of fluorescence that’s detected by a fluorescent light detector; and then sort the cell droplets into Non-fluorescent and Fluorescent bins after being passed through an electric field to determine fluorescence.

With a scatter plot, they proportion of cells expressing fluorescence are quantified.

Use FACS to do Cell Cycle Analysis, to see the proportion of cells in each phase at any one time. Most are in G1 phase so the peak is huge. But in terms of stain intensity, you’ll see that G2 has double the fluorescence as G1 because the DNA in the nucleus has doubled awaiting division.

Question 38

Q

What are some ways to isolate cell Organelles to study them?

Answer

A

Break open their plasma membrane by

Mechanical Homogenization (plunger)
Sonication (ultrasonic waves)
Pressure (force cell through narrow valve)
Non-ionic Detergents (chemicals that disrupt bilayer but still keep the proteins intact)
Hypotonic Solution (burst the cell)

Then Centrifugation
1. Differential — separate homogenate based on mass (the higher the centrifugal speed, even lighter stuff will pellet)
2. Equilibrium Density-Gradient — separate based on density (place homogenate on top of sucrose gradient. With centrifugation help, the organelles will migrate to a sucrose layer that is of equal density to them.)
Then pipette the layer you want.

Question 39

Q

SDS-PAGE

What is is used for?
What is the role of SDS?
What is the basis for separation?

Answer

A

SDS-PAGE used for separating proteins. Use ionic detergent such as SDS in polyacrylamide gel electrophoresis, it’ll denature the protein by disrupting the ionic and H bonds. You place the proteins into wells on the gel, and SDS denatures and binds the hydrophobic side chains of the AAs, making all the protein fragments negative. So when electric field is applied, proteins will move BY SIZE to anode (+).

Question 40

Q

How to detect specific proteins using Western Blotting/Immunoblotting after SDS-PAGE

Answer

A

transfer the proteins from the gel to a membrane so that the first set of Abs can bind well to their proteins. Then wash away excess. Then incubate with enzyme-linked Abs, wash away excess. Then apply the Enhanced Chemiluminescence substrate. Wherever the enzyme and the substrate bind, that’s where the protein is because it’ll glow (which will appear dark on pic)

Question 41

Q

What’s the relationship btwn secretory proteins and ER?

Answer

A

They found that secretory proteins must first enter the lumen of the RER.

Question 42

Q

Explain Cotranslational Translocation

And what is the main key here that they found?

Answer

A

the first few AA of the synthesized peptide will be the signal sequence.
the SRP (signal recognition particle) binds that sequence and guides the whole complex toward the ER membrane.
Here it meet the SRP receptor and binds to it.
GTP binds to the both of them, hydrolyzes to GDP occurs, which opens up the translocon channel. The protein enters the lumen, and the SRP gets recycled to find other sequences.
As the protein enters the lumen, signal peptidase cleaves the signal sequence off.
Stop codon is reached so translation stops
the ribosome leaves
translocon channel closes and protein folds into its conformation in lumen

Question 43

Q

Protein Modification in the ER…

4

Answer

A

specific proteolytic cleavage (seen in cotranslation translocation)
glycosylation (to ensure protein folding, stability and cell adhesion – sugars are sticky)
- this branched 14-residue oligosaccharide structure lives inside ER lumen. This is transferred onto Dolichol (a membrane anchored phospholipid) which links oligo to ER inner membrane. Nascent proteins that are merging into ER have an Asparagine residue that allows for the oligosacc to transfer onto it by oligosaccharyl transferase. Certain proteins may not need all the residues so several glycosidases remove the unwanted sugars.
formation of disulfide bonds (to stabilize)
folding of polypeptide chains
- Chaperone protein BiP binds to protein to prevent bad folding (hydrophobic-hydrophobic interactions)
- the lectin calnexin and calreticulin bind to the oligosacc to prevent their misfolding bec they sticky
- PDI protein breaks or forms disulfide bonds btwn cysteine residues to help fold properly

Question 44

Q

Protein Import in the Mitochondrial Matrix (steps)

Answer

A

cystolic Hsp70 (chap protein) binds the nascent protein to prevent misfolding in cytosol.
The amphipathic mitochondrial matrix targeting sequence located @ N-terminal of protein guides it to the mitochondria where it is recognized by the import receptor on matrix.
This import receptor guides protein to import pore (Tom40 = Translocon of outer mem).
Goes through Tom then goes into Tim44 (Translocon of inner mem).
Matrix Hsp70 binds protein from inside mitochondria and pulls it through (also prevents misfolding in matrix)
Matrix processing protease cleaves targeting sequence.
Protein folds and is active

Question 45

Q

Properties of Golgi Complex

Function of it?

Answer

A

for processing and sorting of proteins (secreted, membrane, lysosomal)
no ribosomes
flattened, disklike cisternae (3 types: cis, medial, trans)
2 flanked network of tubules: CGN – faces RER, and TGN– opposite of RER

Question 46

Q

Budding of Transport Vesicles

Answer

A

COP2 coat proteins associate with the ER membrane via help from membrane associated GTP binding proteins, and the cytoplasmic domain of the cargo membrane protein.
The COP2 proteins associate which promotes the forming and budding of the vesicle, now it is released from the donor ER membrane
Then you want the decoating of the vesicle (otherwise they’ll prevent the vesicle from fusing to Golgi membrane). So the hydrolysis of GTP–>GDP triggers their release.

Question 47

Q

In general terms, what do coat proteins do?

Answer

A

promote budding of vesicles

Question 48

Q

In general terms, what do SNARE proteins do?

Answer

A

promote fusion of vesicles with the target membrane

Question 49

Q

What kind of transport are COP2 vesicles involved in?

Answer

A

anterograde transport (RER to Golgi) – budding

Question 50

Q

What kind of transport are COP1 vesicles involved in?

Answer

A

retrograde transport (cis-Golgi to RER) – fusion/return mechanism

Question 51

Q

Fusion of Vesicles from RER to cis-Golgi

DXE

Answer

A

This is anterograde.

The naked vesicle docks onto Golgi membrane via GTP binding proteins
the v-SNARE protein forms a SNARE complex with the t-SNARE protein, and so now fusion can happen
The membrane associated cargo proteins become part of the Golgi membrane
energy needed to dissociate the SNARE complex, so ATP hydrolysis
Certain cargo proteins use DXE, a sorting signal that helps recruit COP2 coat proteins for other processes later on? (patients with CF can’t do this)

Question 52

Q

What is the Cell Bio relation to Cystic Fibrosis

Answer

A

CF is caused by a mutation in the gene for the protein CFTransmembrane Conductance Regulator (CFTR).
If the mutation occurs, CFTR can’t bind COP2 as well and so anterograde transport is affected (doesn’t work)

Question 53

Q

Vesicle Transport from cis-Golgi to RER

KDEL sorting signal

Answer

A

this is retrograde transport.

A missorted ER-resident protein (got into Golgi by accident) will have a KDEL sequence @ their C-term to send them back.
the cis-Golgi has pH-sensitive KDEL receptors which will recognize the KDEL sequence of the peptide. It will have a high affinity to bind if the pH is low inside the Golgi lumen.
The binding of the KDEL peptide to the KDEL receptor causes the activation of the sorting sequence KKXX (located @ the C-term of the KDEL receptor). The KKXX sequence binds to COP1 coat proteins.
Now the coated COP1 vesicle will travel through the cytosol, shedding COP1 coat proteins, fusing with SNARE proteins, and releasing KDEL ER-resident protein into lumen of ER

Question 54

Q

Which proteins do we know to have the KDEL sequence?

Answer

A

Well you can know that if they’ve been missorted into Golgi, they’ll likely have, but:
Chaperone proteins (ie, BiP)
Lectins
ER-resident proteins

Question 55

Q

Explain the Inclusion-cell Disease

Answer

A

Disease due to absence of a lysosomal enzyme (N-acetylglucophosphotransferase) which results in the accumulation of un-degraded material in the lysosomes. Because M6P isn’t added onto the lysosome, the lysozymes (M6P?) (lysosomal enzymes) are secreted instead. So now glycolipids (N-acetylglucosamine+P) are not degraded and accumulate, producing toxins which can kill the brain cells

Question 56

Q

Protein Glycosylation in Golgi

Answer

A

each Golgi subcompartment has different enzymes. Some have Glycosidases== remove sugars; some have Glycosyltransferases== add sugars.
cis-Golgi: mannosidases
medial-Golgi: GlcNAc-transferases, mannosidase, fucosyltransferase
trans-Golgi: galactosyltransferase, sialyltransferase

Question 57

Q

Trafficking from trans-Golgi network to lysosomes

Answer

A

trans-Golgi network is a sorting station.
In this situation, we’re going to late endosome and then into lysosome, which is facilitated by clathrin and Adaptor Protein (AP)

Targeting soluble proteins to lysosomes requires M6P, a sorting signal which will be added onto the lysosome in the cis-Golgi by GlcNAc-phosphotransferase. M6P created by:
-GlcNAc-phosphotransferase’s Catalytic Site binds the phosphorylated N-acetylglucosamine to Mannose sugar and to lysosomal enzyme/protein to be trafficked. Then phosphodiesterase cleaves off the N-acetylglucosamine to leave the mannose with the phosphate on it==M6P on the protein.
Then M6P gets recognized by the M6P receptor in the trans-Golgi and binds to the protein at pH 6.5. This causes the recruitment of AP coat protein and Clathrin. Membrane pinches off and budding occurs. Decoating happens, and the uncoated transport vesicle fuses with the late endosome. At lower pH (5-5.5 pH) the M6P detaches from its receptor, which allows for the fusion to the lysosome.

Question 58

Q

What is special about Clathrin?

Answer

A

Clathrin forms a Triskelion structure that contributes to the roundness of the membrane

Question 59

Q

How can cells internalize ligands through receptor-mediated endocytosis?
What’s the AP complex for?

Answer

A

this is a SELECTIVE internalization of specific extracellular molecules (ligands)
- when a vesicle starts to form, the protein complex Dynamin associates around its neck, polymerizes and squeezes this clathrin+AP-coated vesicle off with the help of GTP hydrolysis.
The AP complex will recognize the sorting signals of cargo proteins or receptors for the vesicle

Question 60

Q

What are LDLs?

Answer

A

Low-density lipoproteins that are composed of an amphipathic shell (phospholipid monlayer + Apolipoprotein B) & an apolar core (nonpolar, hydrophobic because has lipid, and 88% cholesteryl esters)

Question 61

Q

Explain the binding of an LDL to the LDL receptor

Answer

A

pH dependent binding:
the LDL receptor’s ligand-binding arm binds tightly to the ApoB of LDL @ normal pH (7). This causes the activation of the LDLr’s cytosolic sorting signal @ C-term –> recruitment of coat proteins.
If pH 5-5.5, the Histidine residue in the Beta-propellor domain of LDLr becomes protonated and now binds with high affinity to the - ligand-binding arm on itself

Question 62

Q

Why are late endosomes and lysosomes acidic on the inside?

Answer

A

They have passive chloride channels and ATP-hydrolyzed proton pumps on their membrane. As the H+s get pumped in, anions follow, resulting in acidic lumen.

Question 63

Q

Explain steps of the Endocytic pathway for internalizing LDL

Answer

A

LDLr (in clathrin-coated pits) bind ApoB on LDL
Dynamin helps the pits bud from the membrane–> coated vesicles
vesicles uncoat –> Early endosome (pH 6-6.2). Early Endosome fuses with Late endosome (pH 5). Late endosome too acidic so detachment of LDLr from LDL (by the Histidine residue mechanism)
So now late endosome fuses with lysosome (or goes back up to plasma membrane), and LDL particles are broken down/digested.
LDLr recycles to the cell surface to meet another LDL

Question 64

Q

Why is the internalization of LDL important?

Answer

A

If no internalization of LDL (aka, mutation in LDLr —> no receptor, receptor binds LDL poorly, receptor can’t internalize LDL), then high cholesterol levels in blood is the result == Familial Hypocholesterolemia
This disease can lead to heart attack or stroke before 30.
And then you get lipids flowing to other areas of the body because you’ve got an accumulation of them == Xanthoma

Answer 65

A

This is another type of receptor-mediated endocytosis, in which we are internalizing Iron in the form of Ferrotransferrin (iron+transferrin protein). Ferrotransferrin binds a transferring receptor on PM. —> recruits coat proteins (AP complex) and clathrin, which allows it to bud into vesicle —> de-sheds coat and now early endosome fuses with late endosome —> L endosome has acidic pH so it releases Iron into cytosol, but the transferrin stays bound to the receptor= apotransferrin. —> L endosome goes up to plasma membrane where neutral pH, then transferrin dissociates from receptor.

Difference: here, the receptor-ligand complex remains bound to the L endosome when @ acidic pH

Answer 66

A

A degradative pathway to mediate the turnover of long-lived proteins and abhorrent or excess organelles
- bad cystolic proteins and organelles are degraded by enclosing them in a double membrane vesicle=autophagosome (made from Atg8 and Atg5) which fuses with the lysosome (autophagolysosome) where they’ll be degraded and broken down.
The proteins used + the AAs will be recycled

Answer 67

A

Loss of Atg5 or Atg8: leads to progressive neurodegeneration + early death because there’s an accumulation of protein aggregates that form in brain

Overexpression of Atg5 or Atg8: extends lifespan by suppressing the cognitive deficits that come with age, and suppresses the accumulation of protein aggregates in brain.

Answer 68

A

G1 phase - general growth and metabolism – most time spent here or in G0
Synthesis phase - DNA replication
G2 phase - prep for Mitosis
Mitotic phase - PMATC into 2 identical daughter cells

Answer 69

A

They are terminally differentiated, meaning they are in G0 permanently

Answer 70

A

If the bud is small, it’s still in later phase of G1 (bc duh it has a bud so can’t be early G1).
If the bud is big, it’s in G2
It has a long G1 phase. It produces one small daughter cell and a bigger mom cell

Answer 71

A

Look at cell cycle phase. They’ll have a long G2 and M phase.
Also they grow by elongation, and divide by a septum/wall forming
They produce 2 equal sized cells

Answer 72

A

loss of cdc2 activity (a CDK=cyclin-dependent kinase) would prevent S.pombe from entering into M phase.
So a mutation in cdc2 would mean no mitosis.

Answer 73

A

CDK amount stays the same, but its activity changes because of the degradation and synthesis of the different cyclins at different phases of the cell cycle.
So activity of CDK is dependent on the amount of cyclin

Answer 74

A

Ubiquitin-mediated proteasome-dependent degradation (transcriptional level): cyclins are targeted for destruction by ubiquination at certain phases of cell cycle (which is controlled by SCF ubiquitin ligase):
- ubiquitin conjugating enzyme binds Ubiquitin ligase, which allows the ligase to stick itself to the target protein cyclin. Ubiquitin conj enzyme now sequentially adds Ub (found in all euk cells) to the target protein via polyubiquination, which acts as a signal for cyclin to be degraded by the proteasome.

Answer 75

A

Wee1 would put the phosphate group on CDK, thus inactivating MPF. And you wouldn’t have cdc25 to take it off, so you’ll never go into mitosis, you’ll just stay in G2 where it elongates

Answer 76

A

a kinase which inactivates MPF by phosphorylating CDK

Answer 77

A

a phosphatase which activates MPF by removing the phosphate group that Wee1 put on CDK

Answer 78

A

cdc25 would take off the phosphate group prematurely, and thus MPF would be activated super early and we’d go into mitosis before the cell even got a chance to grow enough, so you’ll get small cells

Answer 79

A

Wee1 must phosphorylate Tyr15 and activate CAK (=an CDK-activating kinase, which phosphorylates Thr161), which will collectively delay the activation of MPF until you introduce cdc25 into the cell, wherein it will remove the phosphate group from Tyr15 and thus activate MPF to go into mitosis at a reasonable time now.

Answer 80

A

You’ll want to do this at anaphase. You use APC/C (=a ubiquitin ligase. anaphase-promoting complex/cyclosome) to degrade cyclin, and thus inactivate MPF. So APC/C recognizes the destruction box on cyclin and so induces polyubiquination of it- where Ub marks the mitotic cyclins for proteolytic degradation in proteasomes –> drops MPF activity in telophase.

Start synthesizing mitotic cyclin is interphase (G1)

Answer 81

A

regulated by degradation of a CDK inhibitor by SCF ubiquination ligase.
- phosphorylate the S-phase inhibitor (that was in place during G1 so as to not prematurely enter S), and will now undergo polyubiquination. This activates the S phase cycllin-complex to initiate S phase (undergo DNA replication)

This same SCF ubiquination ligase will degrade the cyclins at proper time in cycle

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Lectures 1-7 Flashcards

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