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ANAT 212 > ANAT 212 (2) > Flashcards

ANAT 212 (2) Flashcards

1
Q

Causes of cancer theory (3)

A
  1. Humoral theory: excessive black bile caused caused
  2. Infectious disease theory: cancer was infectious
  3. Moder day: viruses, chemical carcinogens and radiation
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2
Q

Hallmarks of Cancer: DNA damaging agents

A
  • radiation
  • viral infection
  • chemical exposure
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3
Q

Hallmarks of Cancer: Sustaining proliferative signaling

A

-Cancer cells do not have to same extracellular signaling
-Have constitutive signaling (can divide without any signaling)

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4
Q

Hallmarks of Cancer: Resisting Cell death

A
  • Mutation pathways can evade apoptosis
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5
Q

Hallmarks of Cancer: Enabling replicative immortality

A
  • Divide forever (non-stop)
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6
Q

Hallmarks of Cancer: Activating invasion & metastasis

A
  • Cancer is able to move from original source
  • Through the lymphatic system
  • ex: breast cancer –> lung/brain
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7
Q

What is a Sarcom?

A
  • Cancer in soft tissue (muscle)
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8
Q

Explain the experiment of Peyton Rouse Avian Sarcoma (chicken/bird cancer)

A
  1. Take sarcoma from chicken
  2. Break it into small pieces
  3. Grind it with sand
  4. Filter it (we don’t want big particles)
  5. Inject it into another chicken
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9
Q

What were the findings of Peyton Rouse’s Avian Sarcoma experiment?

A
  • size of the particle was smaller than the cell itself
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10
Q

What does RSV stand for?

A
  • Rous sarcoma virus
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11
Q

Explain the experiments done to identify RSV.

A
  • on cell culture dish
  • infected cells would infect monolayer
  • would grow on top of each other (focus forming)
  • Carcinogen agent (what caused it) = virus (RSV)
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12
Q

Explain 7 attributes of cell transformation

A
  1. Replicate forever
  2. Altered morphology (round shape)
  3. Loss of contact inhibition
  4. Anchorage-independent growth (soft-agar)
  5. Reduced requirements for GF
  6. Increased glucose transport
  7. Tumorgenicty (can form tumors)
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13
Q

Can Immortalized be transformed cells and vice versa?

A

No:
- transformed cells = Immortalized
- Immortalized cells ≠ transformed

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14
Q

Why does transformed cells high affinity for glucose help us?

A
  • Pet scans
  • Inject the patient with radiolabeled glucose
  • Tumours will take up this glucose
  • Glucose decays and generates positrons
  • can visualize tumors on scans
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15
Q

Is it true that tumor viruses are causative agents for all human cancers?

A

No, viruses are linked to some cancers, but NOT ALL

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16
Q

Explain how RSV retrovirus works

A
  • Retrovirus has a single strand of RNA
  • Uses reverse transcriptase (enzyme) to reverse transcribed RNA into cDNA
  • Integrates itself into host genome
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17
Q

What are 3 important genes in the Viral RNA genome (ALV)?

A
  • Gag gene = core proteins
  • Pol gene = Integrase and RT
  • Env = envelope protein
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18
Q

What is src?

A
  • Sequence driving tumors genesis/transformation
  • Plays an important role in triggering sarcoma formation
  • found in RSV
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19
Q

Is src found in viral or non-virally infected cells?

A
  • Both!
  • Src sequence is present in all normal genomes of the host cell
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20
Q

What is ALV?

A
  • pro-viral DNA
  • integrates next to c-src by chance
  • gets transcribed and packaged into RSV
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21
Q

How did src come to be from an evolutionary aspect?

A
  • ALV was integrated next to c-src by chance
  • Was transcribed/packed into RSV
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22
Q

What is the difference between v-src and c-src?

A

c-src: cellular src gene (proto-oncogene)
v-src: viral src gene (oncogene)

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23
Q

What does expressing high levels of src gene do?

A
  • Doing this in viral infection led to cellular transformation
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24
Q

What is a kinase?

A
  • enzyme that removes high energy phosphate group from ATP and puts it onto a suitable protein substrate
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25
Q

What was the anti-src antibody experiment?

A
  • Src actslike a tyrosine kinase and phosphorylates specififc tyrosine amino acids in subtrates
  • Src can autophosphorylate
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26
Q

Are all oncogenes kinases and vice versa?

A

No, many, but not ALL, oncogenes are kinases

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27
Q

What does PKB influence?

A
  • Phosphorylated downstream substrates either activating or inactivating them
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28
Q

What is an oncogene?

A

Gene that increases selective growth advantage of a cell

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29
Q

What is selective growth advantage?

A

Allows cancer cells to outgrow the surrounding ‘normal’ cells

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30
Q

What is a proto-oncogene?

A

A gene that can become an oncogene because of mutations/overexpression

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31
Q

What is a tumor suppressor?

A
  • Gene that serves as roadblocks = breaks
  • If it is lost/inactive it will lead to an increase in selective growth advantage
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32
Q

Do you need 1 oncogene or the loss of 1 tumor suppressor to cause cancer?

A

NO! It is a build-up of mutations in different oncogenes/losses of tumor suppressors in combination

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33
Q

Do you need virus to form tumors?

A

No! Under the right conditions, you can get it through carcinogens

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34
Q

What was speculated about carcinogen and their possible functioning as mutagens?

A
  • Carcinogens function as mutagens inducing cancer by turning proto-oncogenes into oncogenes
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35
Q

What was NIH 3T3 cell experiment with 3-MC?

A
  • Used NIH 3T3 cells (immortalized fibroblast) because are great at taking up DNA
  • Treat mouse cells with 3-MC (potent carcinogen a component of coal tars)
  • Transfected into small fragment (some contained oncogene) and introduces into host cell = promoting transformation of cells
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36
Q

What are 4 ways to turn a proto-oncogene into an oncogene?

A
  1. Amplification (large number of copies of a small segment)
  2. Insertion/deletion (indel) (of a few nucleotides)
  3. Translocation (Philadelphia chromosome)
  4. Point mutations (single nucleotide substitution)
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37
Q

What is the difference between driver/passenger mutations?

A

Driver: mutation that directly/indirectly confers selective growth advantage to a cell
Passenger: does not confer a selective growth advantage

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38
Q

What is the difference between low/high mutation burden?

A
  • low mutation burden: mutated proteins are low
  • high mutation burden: more mutated proteins = worst
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39
Q

What is karyotyping?

A

-process of pairing and ordering chromosomes

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40
Q

Explain the experiment steps where we found BCR-Abl?

A
  • treat cells with colchicine (drug) that prevents passage from metaphase to anaphase
  • ## stain with Giernsa so you can observe and do karyotyping
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41
Q

What is CML and ALL?

A

CML: Chronic myeloid leukemia
AML: Acute lymphoblastic leukemia

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42
Q

Describe the Philadelphia chromosome.

A
  • translocation between chromosomes 9 and 22
  • generates BCR-Abl (fusion event)
  • found in patients with CML and ALL
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43
Q

What is BCR-Abl?

A

BCR: Breakpoint Cluster Region
Abl: tyrosine kinase (protooncogene that phosphorylates tyrosine amino acid on select tyrosine on downstream substrate)

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44
Q

What does the fusion of BCR-Abl lead to?

A

-Leads to Abl being on crack (unable to turn off)
- Abl has constitutive activity
- inability to be regulated

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45
Q

What is the name of the compound that competes with BCR-Abl and why was it a revolutionary finding?

A
  • Imatinib = compound that killed CML cells but NOT normal ones
  • acted as a competitive inhibitor to BCR-Abl
  • prevent BCR-Abl from phosphorylated because it was bound to the ATP binding site (no more ATP binding)
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46
Q

What is erbB2/Her2?

A
  • Receptor Tyrosine Kinases (RTK)
  • Her2 : GF receptor
  • Utilizes Ras in its process
  • It will insert itself in PM and respond to epidermal GF
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47
Q

What does the dimerization of erbB2/Her2 do?

A

It activates cell proliferation and survival gene expression signaling pathways

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48
Q

Describe the structure of RTKs.

A
  • Has hydrophilic TM proteins
  • Respond to external signals and transmit info into cells through kinase activity
  • Ectodomain: in extracellular space and recognizes/binds ligand
  • Kinase domain: inside cell
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49
Q

How does RTk operate with/without mutations?

A
  1. Without: GF binds and leads to RTK dimerization and activation of kinase domain
  2. With: ligand-independent kinase (doesn’t need GF to be activated)
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50
Q

What happens when erbB2 is amplified? What are the consequences?

A
  • Proliferation, survival and resistance to apoptosis = high
  • off of them surviving with disease-free progression drops
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51
Q

How can we identify erbB2 amplification in the genome?

A

FISH (fluorescence in situ hybridization)
- can observe 2 copies of erbB2 in all our cells (gene amplification)

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52
Q

Describe FISH.

A
  • DNA probe against wanted gene (ex: erbB2)
  • Denature DNA and add fluorescent
  • Hybridize this to on of the copies
  • detect copies of genes in cells
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53
Q

Is it always the case that erbB2 is amplified at the gene level?

A

NO!
- promoters could become hyperactive pumping out a lot of proteins (even without erbB2 amplification in tumor cells)

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54
Q

Why does excessive phosphorylation aid tumor cells?

A
  1. Cellular Signaling Dysregulation
  2. Activation of Oncogenes
  3. Inactivation of Tumor Suppressor Genes
  4. Enhanced Cell Migration and Invasion
  5. resistance to Apoptosis and Chemotherapy
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55
Q

How can we analyze erbB2/HerbB2+ on a protein level (not gene amplification)?

A
  • ICH (immunohistochemistry) utilizes antibody against erbB2 protein
  • If antibody binds (with dye), we can measure the amount of protein based on staining, and its location in the cell
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56
Q

What is a personalized medicine approach that treats erbB2 breast cancer?

A

Traztuzumab (Herceptin)
- Bind extracellular domain and inhibits Her-2 homodimerization (prevents Her-2-mediated signaling)
- CANNOT give this to all breast cancer patients
- wont repond to GF

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57
Q

Is Ras a kinase?

A

NO! It is a G protein (GTP)

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58
Q

Name 5 signaling pathways that Ras provides (downstream pro-signaling pathways)

A
  1. activation of chromatin remodeling mRNA
  2. translational controls of protein synthesis
  3. transcriptional activation in the nucleus
  4. inhibition of apoptosis
  5. cell growth and proliferation
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59
Q

What are the missense mutations in Ras and what does it do?

A
  • Mutations in G12 and 61
  • take Ras from proto-oncogene\e to oncogene
  • Enhances Ras to bind to GTP so it is always active
  • Inactivation of GTPase negative feedback mechanism
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60
Q

How do oncogenes form?

A
  • Upregulation of proto-oncogene expression alters their structure
  • Leads to overly active growth-promoting genes (in cancer cells = activated oncogene)
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61
Q

Are tumor suppressor Genes and oncogenes recessive?

A

NO!
1. TSG: recessive (needs a mutation in both copies for LOF)
2. Oncogene: dominant (1 mutant copy is enough to cancer development)

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62
Q

What are the 2 different ways retinoblastoma can be diagnosed?

A
  • Diagnosed from birth-8 years old
    1. Sporadic form: no family history so single tumor (unilateral = one eye) and can be treated with radiation/surgery)
    2. Familial form: family history so multiple tumors in both eyes (bilateral retinoblastoma) can be treated with radiation/surgery
    • HOWEVER: higher risk of bone cancer (osteosarcoma)
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63
Q

What is the knudson 2-hit hypothesis?

A
  1. Familial form: needs 1 random event (only 1 because already has 1 in genome)
  2. Sporadic form: needs 2 random event
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64
Q

What does Rb do in the cell cycle?

A
  • Acts as gatekeeper on cell cycle control
  • R point = Regulates restriction point where G1 either goes into S phase or G0
  • Too much DNA damage = R point will not let it go to S phase
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65
Q

What is G0 phase in cell cycle?

A
  • Quiescence (reversible) stage
  • no dividing
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66
Q

What are the levels of phosphorylation of Rb depending on its location?

A
  1. In the nucleus: not heavily phosphorylated
  2. In cell cycle: heavily
  3. Stress occurs: does NOT get phosphorylated, instead binds to e2f and prevents it from transcribing genes
  4. Missing Rb: things go into Cell cycle even if they shouldn’t
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67
Q

How can you lose your TSGs?

A
  1. Familial cancers: many inherited mutant TSG
  2. Sporadic: mitotic recombination leads to loss of TSG
    • occurs during G2
    • segregation of chromatid may yield daughters with LOH
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68
Q

When damage is done to the cell, how do we repair chromatid? How does this effect TSG genes?

A
  • repairs through recombination
  • could results in daughter cell having mutations in both chromatids (loss of ‘second hit’ copy from sporadic form)
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69
Q

Can cancer form from the loss of Rb?

A

yes

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70
Q

Can cancer form from the loss of one type of TSG?

A

Additional layers of oncogenes are required

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71
Q

What does LOH stand for?

A

Loss of heterozygosity

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72
Q

Where are TSG found?

A
  • Within chromosomes in regions that are unstable
  • indicates different mutations can lead to the loss of the second allele
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73
Q

What are 4 examples of second hit being lost?

A
  1. Terminal deletion (wt second allele for Rb is lost = becomes hemizygous)
  2. indels (premature stop codon)
  3. Translocation (leads to loss of expression of wt Rb)
  4. Epigenetic silencing (DNA presence is the same, but how active the gene is from being trasncribed changes)
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74
Q

What are some characteristics of activating and silencing domains on chromatin?

A

OFF: heterochromatin (tightly packed, unable to transcribe)
ON: euchromatin (loosely packed)

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75
Q

Sometimes in normal cells, how are tumor cells maintained?

A
  • they are maintained in euchromatic format
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76
Q

How do tumor supressor genes get transcriptionally silenced?

A

through DNA methylation

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77
Q

What is DNMT and what does it do?

A
  • DNA methylation enzyme
  • upregulated in cancer cells
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78
Q

Name 2 tumor suppressor genes and whether they follow the Knudson 2-hit model.

A
  1. p-53 (no = not haplosufficient meaning 1 wt copy is NOT enough)
  2. BRAC1 (yes)
  3. Rb (yes)
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79
Q

How does methylation work? (promoter region? cytosine?)

A
  • cytosine turns into 5 methyl cytosine
  • If promoter region upstream of gene needed to be transcribed = can lead to transcriptional silencing (epigenetic silencing mode)
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80
Q

Is there any way a patient with tumors suffering from loss of TSG because of silencing can get better? If yes, how does this happen?

A
  • give patients drugs to prevent methylation (try to reactivate expression of silenced TSG)
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81
Q

Describe p53, its function, and what leads to its stabilization.

A
  • DNA damage and dysregulated growth signals leads to p53
  • p53 not stable protein until
  • becomes stabilized by becoming homotetramer
  • Transcription factor that halts cell cycle (DNA repair genes, regulators of apoptosis)
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82
Q

What does TSG dominant negative mean? Name an example.

A
  • p53 is an example
  • produces non-functional protein that interferes with normal function
  • only 1 copy is needed (dominant over wildtype)
  • EX: one mutant copy incorporates p53 inhibiting its ability to become a transcription factor
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83
Q

How can we target ‘untargetable’ tumor suppressor gene mutants in cancer cells? Explain process.

A
  • synthetic lethal
  • Gene A and Gene B. Cancer cells = one of the genes if knocked out, so relies on the other, dies, but not normal cells (draw the schematic)
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84
Q

Define synthetic lethal.

A

2 recessive mutants needed to act, alone is unlethal

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85
Q

Benefits of synthetic lethal strategies?

A
  • selective for cancer cell-specific genetic mutations
  • can be applied to any type of cancers mutations (TSG and undruggable tumors)
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86
Q

Difference between BRCA1 and PARP1.

A
  1. BRAC1: TSG homologous recombination
  2. PARP1: base-excision repair
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87
Q

Describe what happens if BRAC1 gets lost, examples, and the consequences.

A
  • If lost, homologous recombination is not available, become es more dependent on other repair mechanisms (PARP1)
  • EX: breast cancer cells have BRCA1 mutated
  • use PARP1 inhibitors to kill cancer cells (used in synthetic lethal matter since BRAC1 will also be mutated in cancer)
  • normal cells stay untouched
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88
Q

Describe Hela cells.

A
  • Named after Henrietta lacks
  • immortalized
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89
Q

Name and describe 2 major obstacles to cellular immortalization.

A
  1. replicative senescence: irreversible halt in cell proliferation with retention of cell viability = metabolically active, but exited from cell cycle forever
  2. crisis: leads to cell death by apoptosis
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90
Q

Explain replicative senescence and hayflick limit.

A
  • replicative senescence: process of getting old
  • limit: # of times human cell divide until cell division stops (enters senescence)
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91
Q

Discuss telomeres and how they relate to senescence. (bypass?)

A
  • senescence is triggered by telomere shortening (can be bypassed by TSG mutations)
  • after bypass, cell undergo crisis (chromosome fuse leading to apoptosis) (telomeres get short = p53 independent)
  • causes chromosomes to fuse/break
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92
Q

Describe breakage fusion bridge cycling.

A
  • chromatid ends without telomores fuse:
  • Chromosome is formed due to abnormal chromosome breakage and fusion
  • snap at anaphase and cause genetic instabilities
  • kill some cells (mitotic arrest)
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93
Q

Describe escaping crisis.

A
  • a way of immortalizing cells
  • cell overexpresses enzyme that keep telomere long
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94
Q

Describe hyperplasia

A
  • increases in NUMBER of cells
  • Occurs in cells withe the capacity to divide
  • occurs in liver regenration and epithelial cells of pregnant ladies
  • once stimuli is gone, it is reversed
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95
Q

Describe hypertrophy.

A
  • increase in SIZE of cell
  • no ability of dividing
  • occurs in skeletal when working out, in cardiac muscle
  • once stimuli is gone, it is reversed
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96
Q

Describe neoplasia

A
  • new growth (tumours)
  • excessive proliferation of cells
  • irreservsible
  • arises from genetic alteration
  • can be benign (not cancer) or malignant (cancer)
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97
Q

Compare benign and malignant tumors.

A

benign:
- no invasion of BM or neighboring cells
- mostly harmless (unless high hormone levels are sent to specific places like brain)
malignant:
- invasion of BM and neighbors
- can metastasis (travel from origin)
- 90% of cancer death are from metastasis
- cells who took up mesenchymal transition infiltrate lymphatic system and invade other tissues

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98
Q

Describe dysplasia.

A
  • cells look abnormal
  • not cancerous (pre-cancerous)
  • can be mild, moderate or severe
  • could result in cancer or not
  • reversible
  • results from genetic alterations
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99
Q

What is the difference between the theoretical vs reality of cells forming tumors?

A

Theoritical: exponential growth
Reality: not all cells survive as they divide (some die through apoptosis)
- cancers develop over many decades - age is a big factor

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100
Q

Do cancers show up right when the original carcinogen happens?

A

NO! tumors take many years to pop up from original carcinogenic on original cells
- we see lag in data

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101
Q

Discuss colorectal cancer and why they are the best models to study metastasis and progress

A
  • colon and small intestine covered by epithelia cells (tight junction, thin layers, fast turnover)
  • the villi: BM composed of ECM allowing epithelial cells to grow
  • Epithelial layer: site of most pathological changes associated with the development of colon carcinoma
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102
Q

Difference between carcinoma and adenoma. What about adeno carcinoma?

A

Adeno carcinoma: derived from intestinal epithelial cells
- occurs due to loss of p53
carcinoma: malignant, no BM breach and cannot metastasise (lacks certain malignant characteristics) (pre-malignant/cancerous)
adenoma: benign, no Bm breach
polyp:
- slow growth
- no potential of spreading

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103
Q

What is EMT? Describe the process.

A

Epithelial to mesenchymal transition
- epithelial cells acquire mesenchymal traits (loosely organized, fibroblast-like morphology, increased invading abilities)
- also acquire behavior following the downregulation of epithelial features (form barriers, very organized)
- occurs during embryogenesis, wound healing and cancer

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104
Q

Name 4 core EMT changes.

A
  1. cytoskeletal remodeling
  2. cell-cell adhesions weakening
  3. acquisition of cell mobility (moving from one cell to another)
  4. BM invasion
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105
Q

Explain the invasion-metastasis cascade. What is the difference between Intravasation and Extravasation?

A
  1. localized invasion (in situ carcinoma cells break BM)
  2. Intravasation into lymphatic system and spread
  3. locate to another tissue tissue forming metastatic tumors
  4. Extravasation: where the cells land and set up metastasis (require extra traits depending on where they end up)
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106
Q

What is MET?

A
  • mesenchymal to epithelial transition
  • acquire polarity back (apical-basal), cell-cell adhesions, reorganized cytoskeleton
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107
Q

What is an example of primary tumors and metastatic destination (Mind map)?

A

prostate: brain, lung, liver, bone marrow
pancreas: lung and liver
breast: all
colon : lung, liver and bone

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108
Q

Name the 6 major tissue types

A
  1. epithelial
  2. connective
  3. bone
  4. nerve
  5. lymphatic
  6. muscle
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109
Q

Name the 3 cell junctions. Also name further examples of each.

A
  1. occluding junction (tight junctions (vertebras) and separate junctions (intervertebral))
  2. anchoring junction (1. actin filaments (cell-cell (adhesions) and cell-matrix (focal)) 2. intermediate filaments (cell-cell (desmosome) and cell-matrix (hemidesmosome))
    3 communications junctions (gap junctions)
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110
Q

Define the 3 cell junctions.

A

occluding: seal cells together in an epithelium, no leakage from any side
anchoring: attach cells to neighbors or ECM
communicating: mediate passage of signal from one cell to another

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111
Q

Describe the process of a molecule passing through tight junctions. Discuss the 2 sets of TM protein, if they mix, etc.

A
  1. seperation of the proteins allows for vectorial transfer of nutrients across thee epithelium
  2. tight junctions are located under microvilli
  3. sodium driven glucose symport (against gradients to BM)
  4. glucose carriers at BM take the glucose
  5. This travel is done passively (no ATP)
  6. both glucose and sodium bind the symport
    function: protein localization, prevents backflow and acts as diffusion barrier for PM
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112
Q

Discuss light and electron microscope: difference, specifics, etc.

A

wavelength (up) velocity (down)
- accelerating volatge: 100,000 V
- wavelength = 0.004 nm
- practical resolving power for biological specimen = 1 nm
- source of e= cathode
- acceleration : e- through anode
- ultralow pressure vacuum
- magnetic coil focus beam (glass lenses for LM)
- tissues are not dense we need staining (osmium tetroxide= bind to lipid bilayer and proteins)
- specimen needs to be thin because e- have limiting penetration power

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113
Q

explain tracer molecule and tight junction

A
  • small, e- dense and extracellular
  • no passage through tight junction (no matter what direction)
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114
Q

explain ultralow pressure vaccuum in microscope

A

e- will scatter by collision with air = try to keep the air as minimal as possible

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115
Q

explain freeze-fracture electron micrograph and e and p face.

A
  • rapid freeze of cell where PM is cleaved down the fracture plane
  • E face: inner face of the outer monolayer
  • P layer = inner face of the inner leaflet (complementary face)
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116
Q

Discuss the 2 major proteins in sealing strands.

A
  1. Claudin (most important)
    - 20-27 dKa
    - Claudia-16 mutation = kidney, excessive loss of MG2+ from urine to blood
  2. Occludin
    - bigger, 67 kDa
    - 2 isoform produced by alternative splicing
    - localization of occlusion done by phosphorylation
    BOTH HAVE:
    - 4 alpha helical TM helices (2 extracellular loops, c-terminus located inside the cell)
    - tight interactions with each other
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117
Q

Give an example of tight junctions and how it relates to the anchorage to the cytoskeleton. Draw it out.

A

ZO: zonula occludens
- intracellular connection to the cytoskeleton
- tight junction complezes connect to actin filament
DRAWING:
- sealing strands of tight junction proteins hold adjacent PM
- strands composed of tM proteins making contact accross the intercellular space and created seal

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118
Q

Explain how to go from protein mass to amino acid to # of nucleotide.

A

protein mass: 67 kDa.
1. divide by 100 to get amino acid (100 being the mass of amino acid)
2. multiply by 3 (because 3 nucleotide make up 1 amino acids)

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119
Q

Name the 3 types of cytoskeletal fibers

A
  1. Actin filament (actin)
  2. intermediate filament (desmin, keratin and vimentin)
  3. microtubules (tubulin, not apart of cell junctions)
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120
Q

Name and explain a tight-junction-associated disease.

A

CLDN-14 (claudin 14 mutation)
- nonsyndromic deafness
- we have epithelial tissue in our ears important for hearing, so mutation could lead to deafness

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121
Q

Where would we find anchoring junctions? Describe their structure (strong?)

A
  • intestinal
  • skin
  • other epitehlial cells
  • they are weak and flimsy, so anchoring junctions provide support and stability
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122
Q

Name 2 principal classes of anchoring junctions. Describe their specifics (draw structure)

A
  1. TM adhesions proteins
    - cell-cell: interact with themselves (hemidesmosome (IF) (cadherins) and adherent junctions (actin))
    - cell-matrix: interact with matrix (hemidesmosome (integrins (IF)) and focal adhesions (a))
  2. Intracellular anchor proteins
    - connect to actin, to TM adhesions proteins
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123
Q

Describe cadherins and specify what class of junctions they belong to.

A
  • adherens junctions
  • homophilic interacts with itself
  • homodimers
  • calcium-dependent
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124
Q

Give examples of intracellular (adapter) proteins. Give the 3 different types of them.

A
  • catenin
  • vinculin
    3 types:
    1. p-120
    2. beta-catenin
    3. alpha-catenin (longer and interacts with vinculin)
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125
Q

Where is the adhesions belt and what is it?

A
  • located below tight junctions
  • is made up of adherens junctions (cadherins)
  • could also be referred to as zonula adherens
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126
Q

Explain the process of the neural tube.

A
  • epithelial cells
  • invagination occurs
  • pinching off gives rise to the neural tube
  • occurs through contractibility forces from the actin-myosin II system (ATP dependent)
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127
Q

Describe the actin-myosin II system.

A
  • Made up of 2 heavy chains
  • 4 myosin light chain
  • myosin dimerizes and self-assembles (forms bipolar filaments)
  • Bipolar filaments interact with actin filaments (utilize ATP to pull them together)
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128
Q

Describe desmosomes, what they interact with, and specific proteins associated with them. Draw it out. (discuss adhesion proteins, not adapter ones)

A
  • desmosome interact ith intermediate filaments in a sideway manner
  • cadherins found in desmosome include: (adhesion TM proteins)
    1. desmoglein
    2. desmocollin
  • Epithelial cells = keratin filaments
  • cardiac cells = desmin filaments
129
Q

Explain a human disease that is related to desmoglein.

A
  • pemphigous vulgaris: affects skin and mucous membranes
  • autoimmune disease that attacks desmoglein rendering desmosome weaker
  • results in internal and external blisters
130
Q

Discuss adapter proteins involves in desmosome.

A
  • Plakogoblin: globular strcuture that connect to cadherins and desmoplakin
  • desmoplakin: connects plakoglobins to intermediate filaments
    • forms a dimer that is a coiled-coiled region that holds subunits together
131
Q

Describe the coiled-coiled region found in desmosome.

A
  • takes 7 amino acids to make 2 turns
  • Amini acid A and D (hydrophilic) every 4 amino acids, are on the inside
132
Q

Describe focal adhesions and specify what junction family they belong to. Draw it out.

A
  • belong to anchoring junctions
  • connects cell to ECM
  • use integrins (TM protein) which forms heterodimer (alpha and beta)
  • beta subunit is attached to actin through anchoring proteins (talin, alpha-actinin and filamin)
133
Q

Describe an experiment done to visualize actin and vinculin and discuss the results/

A

WT:
- actin: seen that it runs end to end
- vinculin: peripheral
MUTATIONS: alpha 2 - beta 1 (connect to collagen 1)
- the days are messed up
- shows that the connection of alpha 2 to beta 1 is crucial for the connection to collagen 1

134
Q

Describe the hemidesmosome and the proteins related to it.

A
  • cell and ECM (IF to ECM)
  • integrin: alpha 6 and beta 4
  • anchoring protein: plectin
  • connect to IF (ex: keratin)
  • BM: always made up of laminin and collagen type IV
135
Q

The BM is anchored to deeper layers of the skin by which filaments?

A

Anchoring filaments named fibrils (collagen type 7)

136
Q

Name 5 mutations that stand for EB and have to do with different levels of tissues. Describe severity.

A
  1. keratin 5 and 14 (simplex)
  2. plectin (hemidesmosome)
  3. integrin alpha 6 - beta 4 (hemi)
  4. laminin 5 (junctional)
  5. type 7 collagen (dystrophic)
    Small to big severity
    - 7 collagen is worst because everything sits on it
137
Q

Describe Gap junctions and what type of molecule can pass through it.

A

Gap junctions are a form of communication junctions
- small (< 1000 dalton) vitamin, ATP, sugars, amino acids, water and proteins (electric coupling with Ph) diffuse freely
- bigger (> 1000 dalton) proteins, enzyme, macromolecules and RNA cannot pass.

138
Q

Describe what channels are needed in gap junctions for bigger molecules to pass.

A

Connexins: 4 TM proteins (N = in cell, C= cytosol)
Connexons: 6 connexins
12 connexins = 2 connexions = intracellular Channel
- continuous aqueous channel
- can be hetero and homo typic (channel) or meric for (connexons)
Gap= 2-4nm

139
Q

Discuss Gap junctions regulations.

A
  • High calcium / low pH (inside cell)= closed
  • Low calcium / high pH = open
  • Extracellular: very high levels of C2+
  • calcium is a second messenger (receives signal outside cell and relates it to inside) and is tightly regulates
  • when cell dies: ca2+ rushes inside cell, closing gap junction so it doesn’t affect its neighbors
140
Q

What is calmodulin

A

calcium binding protein that binds to gap junctions and calcium

141
Q

Name 5 permanent cell-cell junctions and name the complex that 3 of them form.

A
  1. tight junctions
  2. adherent junctions
  3. desmosome
    (form junctional complex)
  4. gap junctions
  5. hemidesmosome
142
Q

Do epithelial cells form focal adhesions?

A

NO! epithelial cells are too organized (focal = disorganized = smooth muscles cells and fibroblast)

143
Q

Describe difference between cell junctions vs cell adhesions.

A

Cell junctions (EM):
- “permanent” anchorage
- stability
- communication
Cell adhesion (functional assays)
- transient anchorage
- cell-cell recognition
- typically before cell junctions are established
- partially overlapping molecules (cadherins, integrins)

144
Q

Describe the process of neural tube in the context of cell adhesions. Draw it out

A
  • cells crawl out of space and migrate down to differentiate into nerve cells and peripheral ganglia
  • cell-cell recognition MUST occur to make sure everything happens properly
  • CAM mediates cell adhesions
  • CAM= cell adhesion molecules
145
Q

Name 2 types of CAM.

A

E - cadherins (ectoderm)
N-cadherins (neural tube)

146
Q

Describe the cell-dissociation experiments (embryonic chicken tissue that is flimsy)

A
  • dissociates easy
  • uses trypsin to digest (protease)
  • EDTA (Ethylenediamine-tetra acetic acid) will chelate (bind to metal ions (Ca2+)
  • if u mix dissociated cells they will resemble their original structure (liver and retina)
147
Q

Describe cadherin dependent cell sorting.

A
  • if you mix E and N cahderins they will sort each other out (similar segregation for cells expressing high and low levels of the same cadherins)
  • L cells do not express cadherins
  • Use chaderins as a blank sheet when overexpressing them so artificially see the cadherins that we want
148
Q

Name the 8 cadherin superfamilies. Briefly describe if needed.

A
  • classical cadherin (E): cadherin domain (independent structures)
  • fat-like cadherins (longer onextracellular space)
  • 7 pass T cadherins (flamingo) (cadherins with G protein)
  • protein kinase cadherins (Ret): attached to kinase
  • desmosomal cadherin (desmocollin)
  • proto cadherins = in the CNS
  • T-cadherins = EXCEPTION = does not have TM but is anchored by GPI that is anchored to PM
149
Q

describe the structure of classical cadherin.

A
  • 2-3 calcium binding site
  • without calcium, structure changes
  • not a loose structure
  • forms dimers and large oligomers
150
Q

You have cells on the plate, how do you get them off?

A

add EDTA to remove/chelate calcium to weaken molecules and we can also add protease (trypsin) to detach the cells from the support

151
Q

What is the function of calcium in cadherins

A
  • provide rigidity (stiffness)
  • EDTA removes calcium leaving it flimsy and gets degrades
  • getting calcium back on cadherin makes it more stable and it able to form a homodimer
  • 1 mM Ca2+ concentration is good enough to saturate calcium binding sites to allow both homodimer to bind
152
Q

Difference between affinity and avidity.

A
  • Affinity – describes the interaction of 2 components – relatively low
    for cadherins
  • Avidity- accumulated strength of multiple affinities – high for
    cadherins
153
Q

Explain how cadherin interaction works (describe affinity/avididity)

A

Individual Cadherins bind with low affinity but high avidity
- these interactions happen on the N-terminus
- cadherins are long, so gaps are long
- strong attachment results from many interactions

154
Q

When looking at a mouse embryo, what do you notice at a certain stage when E-cadherin is expressed?

A
  • at around 16 cells stage we see it
  • more strength and close adherence
155
Q

Describe role that Cadherin plays in the CNS. Discuss non-classical cadherins also.

A
  • makes sure the right connections are made in the developing neurons
  • non-classical = protochaderins
  • clusters of genome that provide more variability for cadherins
  • Red exons preceded its own promoter
  • if transcription starts at V8 it will be the only exons there
  • V8 exons code for the entire extracellular portion of cadherin
156
Q

What is Rac1, GDI, PI3K and GEF.

A

1.Rac1: GTP binding signaling molecule
2. GDI: GDP dissociation inhibitors , which interact with GDP-bound small GTPases,
inhibit the exchange of GDP for GTP, and sequester the small GTPases into the cytosol
3. PI3K: Phosphoinositide 3-kinase
4. GEF: Guanine nucleotide exchange factors (GEFs), which promote the exchange of
GDP for GTP

157
Q

Explain the activation of Rac1

A

kept inactive bound to GDP and GDI
- gets releases and gets located near cadherin
- cadherin touch and activate PI3K which activates GEF
- Rac1 activated and bound to GTP promotes actin self assembly
- creates a forces that pushes on cell membranes and its neighbours
- promotes cell-cell adhesions

158
Q

Describe selectins and name 3 examples of them.

A
  • calcium-dependent cell adhesions
  • has a lectin domain: binds to sugars not proteins
    EX:
  • L selectins (lymphocytes)
  • P selectins (platelet, endothelial cells)
  • E selectins (activated endothelial cells)
159
Q

Describe function of selectins during an inflammation event.

A

Flow of blood is too fast
- endothelial cells (in artery wall) express selectins and lymphocytes express oligosaccharides
- have a weak interactions between them
- force of flow makes lymphocytes roll helping it slow down (rolling = selectin-dependent)
- integrin-dependent mechanism are activated and bind cell to endothelial layer

160
Q

Discuss N-CAM and its different forms, extracellular domains and proteins.

A

-cell adhesion molecule (CAM)
- - Calcium independent and homophilic interactions between 2 N-
CAMS
- 20 different forms (due to alternative splicing)
- co-expression of cadherins and Ig-like CAMS
- N-CAM fine tunes interactions of cadherins during development and regeneration
- extracellular domain has di-sulfide bonds (good for support)
- Protein needed to cystein bond= protein disulfide isomerase

161
Q

Name 3 Ig-like CAMs.

A
  1. Neural cell adhesion molecules (N-CAM)
  2. Intercellular adhesion molecules (I-CAM)
  3. Vascular cell adhesion molecules (V-CAM)
162
Q

Discuss the presence (or lack there of) of interns in vertebra’s and procaryotes). Name a worm example and explain.

A
  • no interns ins procaryotes (bacteria), plants, fungi or single cell organism
  • vertebrates have 18 alpha subunits and 8 beta subunits (theoretically 18*8=144 possibilities but its not true, limitations of which alpha can bind to which beta)
  • Caenorhabditis elegans: two alpha subunits and
    one beta subunit => form two integrins (each alpha forms a heterodimer with the beta
    subunit)
163
Q

Discuss the mammalian integrant receptor family.

A
  • 24 integrins heterodimers (not 144 lol)
  • integrins participate in cell aggregation, cell-cell adhesions and cell-matrix adhesions.
164
Q

Describe integrins heterodimer structure

A
  • small intracellular c-terminus
  • small TM region
  • big extracellular domain (N) big head + big leg part
  • requires cation
  • type 1 TM (n outside c inside)
  • integrins are connected via intracellular anchor proteins to actin filament (except: a6b4 – intermediate filaments)
  • Alpha subunit has Furin (protease) cleavage site
  • it is held together by a disulfide bond
165
Q

Name and explain a integrin-binding sequence.

A

RGD= arginine (big positive), glycine (small), aspartic (negative) (could be replaces by glutamine (E)
- this sequence can be found: fibronectin, fibrinogen and some collagen

166
Q

Explain some key concepts about integrant ligand binding site (alpha or beta binding first?) Give an example.

A
  • FIRST RGD (aspartic acid specifically) makes contact with calcium ions bound to beta subunit
  • SECOND synergy binds to alpha subunit (how it binds to integrin)
  • when both are bound, better binding occurs
    EX: fibronectin
  • FN10: RGD
  • FN9: singer
167
Q

Discuss integrant activation, specifically about cell aggregation.

A
  • alpha 2B - Beta 3 integrant involved in cell aggregation (wound healing)
  • mediates blood platelets aggregation (blood clots to stop bleeding)
  • high regulation = too much bad (stroke)
    HOW IT HAPPENS:
  • exposure to collagen IV or thrombin (outside blood vessels)
  • activates integrant (a2B-b3) which will bind fibrinogen (protein for blood clot formation)
168
Q

Name a disease associated with blood clot problems.

A

Glanzmann thrombasthenia: mutations in beta3 integrins (bleeding disorder)

169
Q

Are integrins always constitutively active? Explain 2 processes.

A

NO! can be unactivte on cell surface
-unbound integrins diffuse freely in the PM
activation:
1. outside-in
2. inside-out
Important for:
1. cell aggregation (platelets)
2. cell-cell adhesions (leucocytes and inflammation)

170
Q

Explain Outside in process.

A
  1. ligand is missing, it looks bent = adding ligand changes conformation to an upright, active one
  2. Binding to the bent inactive conformation (weak interaction)
  3. straightening of alpha and beta chains, extracellular ligand will bind much better
  4. conformational change in beta subunit head domain
  5. separation of the entire alpha and beta subunits (essential for activation)
  6. active integrin
171
Q

Explain inside out process.

A
  • inactive = salt bridge between arginine on alpha and aspartic acid on beta (ionic non-covalent interaction between arginine is positive, aspartic negative)
  • talin binding region on beta subunit
  • Talin needs to be activated first before activating integrins (inactive = cannot bind to region and active integrins)
    1. CALPAIN: protease and can cleave the head domain of talin (activate it) and can bind to beta subunit
      -head domain would bind to talin binding site region and disengage salt bridge
  • 2 subunit move apart and become active
    2. PI4,5P-2: causes conformational change, opens up talin structure
  • makes head domain available like before (not separated) salt bridge same thing as before
172
Q

define disintegrins and specify two ways it acts as an inhibitor.

A
  • small proteins (400-100 aa long) with high homology to each other
  • found in snake venom proteins that contain cyetin residues and RGD
  • 40 identified
  • black function of some integrins
    1. act as competitive inhibitor for integrin-fibrin interaction and block blood coagulation (anti-coagulation agents, aIIbb3 = reduces heart attacks)
    2. inhibits angiogenesis (blood vessel formation) in cancers (cancers need blood supply to provide nutrients)
173
Q

Similarities between disintegrins loops and fibronectin loops

A
  • disintegrins will competitively inhibit the binding od fibronectin
  • disintegrin: has loops with RGD reading to bind to integrins
174
Q

where do integrins form clusters? describe its affinity/avidity

A
  • low to moderate affinity
  • very high avidity
  • ligand binds to integrins promotes the lateral diffusion and redistribution of integrins to focal complexes, where they become clustered.
175
Q

Explain fibronectin assembly and how it stains, is there co-stainers?

A
  • secretes in the extracellular space as dimers and curled up (not active)
  • bind integrin lapha 5 beta 1 which connect to actin filament contracting with the help of myosin
  • opens up firbronecting structure and opening self-assembly sites
  • fibronectin runs on top of cell
  • contains with intracellular actin filaments (are dependent to each other when using cytochalasin which disrupts actin filaments)
176
Q

describe structure of collagen. (general, no sequence) what are the two groups?

A
  • 3 polypeptide alpha chains (not alpha helix)
  • 46 collagens genes make 26 collagen types
    1. fibril-forming
    2. sheet-forming
  • exist as hetero or homo trimers
  • forms aggregates
177
Q

describe sequence of collagen in structure.

A

Gly-X-Y (glycine always third and in the middle, most likely to have mutations) (x is proline) 9y is hydroxyproline)
- hydrogen bonds are mediated by X and Y

178
Q

explain collagen synthesis steps (9)

A
  1. pro-peptide synthesized come into ER
  2. hydroxylation of selected prolines (by prolyl hydroxylase and lysyl hydroxylase)
  3. glycocylation of selected hydroxylysine (monosaccharide = galactose) (disaccharide = glucose)
  4. self assembly of 3 pro-alpha chains (have recognition sites for pro-peptides on c-terminus) and forms helix
    pro peptides are kept for 2 reasons:
  5. prevent formation of big collagen fibres inside
  6. to make sure that the intracellular helix formation occurs correctly
  7. formation of helix (from c to n)
    deep invagination:
  8. secretion
  9. cleavage of pro peptides in ECM
  10. self-assembly into collagen fibrils
  11. aggregation into collagen fibres
179
Q

discuss cleavage proteins/enzyme used to cut off pro peptides in ECM (collagen synthesis)

A

Procollagen N-proteinase
n terminus: ADAMTS -2-3-14
Procollagen C-proteinase
c terminus: Tolloid family (BMP-1, mTLD, TLL-1)

180
Q

discuss overlapping issues with formation of fibrillar colllagens. explain each part.

A
  • entropy driven self-assembly process
  • Stagger: each triple helix stack up but not in perfect parallel (stagger)
  • 5 molecules = collagen microfibril
    BANDS:
  • hole zone (positive and negative line up = looks darker because it has more osmium tetroxide staining)
  • overlap zone (nothing, lighter)
181
Q

are collagen and bacteria visible with EM?

A

YESSIR ONLY FOR COLLAGEN
bacteria: LM

182
Q

expand on the specific uses of each hydroxylase.

A
  • Lysyl hydroxylase:
    1. Important for glycosylation
    2. Important for crosslinking
  • Prolyl hydroxylase:
    1. Intermolecular hydrogen bonds
183
Q

explain the process of enzymatic mechanism of prolyl hydroxylase (drawing)

A

reactants: O2, prolyl residue and ketoglutarate
co-factors:
- Fe2+
- ascorbate (vitamin c)
- diozygenase (prolyl hydroxylase)
product:
- c02, succinate and 1-hydroxyl prolyl residue
IF NO COLLAGEN AVAILABLE:
- Oxidizing iron to Fe3+ making it unactive
- Vitamin C (ascorbate) helps bring Fe3+ to Fe2+

184
Q

name a defect in collagen hydroxylation and how it was solved.

A

scurvy: lack of vitamin C (slow wound healing, teeth falling apart, bones breaking)
- fixed with Sauerkraut – keeps long (fermented), rich in vitamin C and iron

185
Q

Explain LOX. explain the mechanism as well.

A

lysyl oxidase
- work in extracellular space and introduce cross-links into collagen fibers
- 5 differente enzyme: LOX and 4 LOX-like enzymes
MECHANSIM:
- lysine side chain
- deanimation
- add oxygen
- makes aldehyde: (hydroxyl)allysine which is very reactive so next step is non-enzymatic (aldol condensation)
- forms cross-link

186
Q

summarise Prolyl hydroxylase, Lysyl hydroxylase, N- and C-Pro-peptidases and Lysyl oxidase

A

prolyl: ER/Golgi, Ca2+ and Fe2+ dependent
lysyl: ER/Golgi, Ca2+ and Fe2+ dependent
propeptides: extracellular, Zn2+ dependent
lysyl oxidase: extracellular
Cu2+ dependent (copper)

187
Q

name a collagen defect

A

Ehlers Danlos Syndrome
Joint hypermobility, stretchy skin, scar formation is incomplete and cardiovascular

188
Q

Name 6 examples of collagen problems you can have.

A
  1. Classicaltype
    (Collagen type V defect, in a1 chain)
  2. Hypermobility type
    (tenascin XB defect) (collagen fibrillogenesis) 3. Vascular type
    (Collagen type III) (aortic aneurysm)
  3. Kyphoscoliosistype
    (Lysyl hydroxylase-1 deficiency)
  4. Arthrochalasiatype
    (Collagen type I)
  5. Dermatosparaxistype
    (ADAMTS-2 defect)
189
Q

what is metabolism. give definition, dependencies.

A
  • Process through which living systems acquire and utilize the free energy they need to
    carry out their various functions
  • ATP-dependent
  • constant ATP/energy consumption
  • fat cells=stores energy in forms of fat
  • Some energy is secreted (organic waste) or used to produce work (movement) or heat
190
Q

what is ∆𝐺°’

A

free energy available in the glucose molecule under standard condition

191
Q

difference in glucose and palmitate (fatty acids) in their ∆𝐺°’ numbers

A
  • Glucose: burn a single molecule (delta=big number) (glucose is stored as glycogen)
  • Palmitate (fatty acid): oxidize and burning it (bigger number) (fat stores as triglycerides)
  • storing energy as fat is a very efficient way to store energy
192
Q

what does ATP drive?

A

unfavourable reactions
- ∆𝐺°’ = -32.3 kj/mol
- atp is the energy currency of the cell

193
Q

is ATP energetic itself?

A

no, The ability to cleave the last phosphate from ATP is easy, but there is nothing inherently energetic in the ATP molecule itself (it’s the ratio to ATP to ADP that gives it its ability to drive unfavorable reaction)

194
Q

is there more free energy in glucose and fat or ATP?

A

glucose and fat
- oxidizing glucose to make more ATP (tons of sugar to make ATP)

195
Q

difference between catabolism and anabolism

A

Catabolism : complex to simple (glycogen to glucose) - production of ATP from bonds breaking
- Exergonic
Anabolism: simple to complex (fatty acid to triglycerides) - Endergonic (requires energy input)

196
Q

What are the 3 steps of metabolism?

A
  1. Ccomplex (Proteins, nucleic acids and polysaccharides)
    • Monomers (amino acids, nucleotides, sugars, fatty acids and glycerol)
    • metabolic intermediate (pyruvate, acetyl CoA and citric acid cycle)
  3. simple small comelecules (H20, C02, Nh3)
197
Q

when does oxidation occur and what does it do?

A

occurs in glycolysis and it extract energy from complex molecules

198
Q

In general, after oxidation, the cell has to deal with the resulting elections by putting them where?

A

on an acceptor molecules like (NAD), lactate for glycolysis and oxygen for the electron transmission

199
Q

Can the metabolic intermediates in glycolysis leave or must stay?

A
  • they can get syphoned off into another metabolic pathway, it is not linear but like branches!
200
Q

Name of the reverse mechanism of glycolysis

A

Gluconeogenesis

201
Q

describe futile cycle. describe our metabolite activity can act as inhibitor.

A
  • add ATP to acetyl-CoA we can make fatty acids (and opposite)
  • happens simultaneously which means wasteful consumption of energy without any net production
  • normally Metabolite that activities one side normally inhibit the other, so separating both arms make it easy to regulate
  • Allosteric regulation: stops futile cycles from happening
202
Q

What does glycolysis mean

A

splitting sugar

203
Q

define Substrate level Phosphorylation

A

metabolites have higher phosphorylation potential than ATP, can phosphorylate ADP directly

204
Q

where is energy stored in mitochondria

A

energy is not store on an intermediate (metabolite) but in the electrochemical gradient

205
Q

describe pathway that favors oxidation of glucose to put phosphate on metabolites

A

have lower ∆𝑮°! than ATP (lower G means favorable=good)

206
Q

describe which tissues synthesize versus use glucose as an energy source

A

synthesize: liver, kidneys cortex
energy source: brain and
nervous tissue, muscle, kidney medulla, erythrocytes and testes

207
Q

What are 2 phases of the glycolysis pathway

A

energy investment and generation stage

208
Q

what are outputs of the glycolysis

A

1 glucose + 2 ATP = 4 ATP, 2 NADH and 2 pyruvate (only 2 ATP at the end cuz we invest 2)

209
Q

How many lactate molecules are made from 1 glucose?

A

2

210
Q

describe what happens at the end of glycolysis and how we keep it going?

A
  • NADH is an inhibitor of glycolysis
  • Must convert it back to NAD to keep glycolysis moving : lactate dehydrogenase reaction (take pyruvate and NAHD and make into lactase, regenerating NAD+) (favorable)
211
Q

What is the first step of glycolysis

A

Hexokinase: first investment of ATP
- makes glucose into G6P
- does this to trap glucose (brought in my transporter, phosphorylation traps it in cell)
- hexokinase has very high affinity of glucose
HOWEVER: glucokinase (isoform hexokinase in the liver) has a poor affinity for glucose
- this is because liver produces glucose, so no need to trap it

212
Q

Describe hexokinase in terms of binding sites and equations

A
  • Glucose + phosphate (Pi) ⇌ G6P + H2O (∆𝑮°’ = +13.8 kJ/mol)(unfavorable)
  • ATP + H2O ⇌ ADP + Pi (∆𝑮°’=-32.2 kJ/mol) (favorable)
  • Adding them up = around -19 kJ/mol, making G6P
    has a binding site for both glucose and ATP (transferring
    phosphate from ATP to glucose)
213
Q

Describe step 3 of glycolysis

A
  • Phosphofructokinase = PFK
  • second investment of ATP
  • Converting F6P to FBP
  • Taking a phosphorylated molecule and double
    phosphorylating
214
Q

Describe step 4/5 of glycolysis

A
  • Aldolase/Cleavage
  • splitting FBP into 2 triodes (GAP and DHAP)
  • both are in equilibrium= when you make DHAP, you get GAP right away
215
Q

describe step 6 of glycolysis

A
  • GAPDH/Oxidation and phosphorylation
  • Takes NAD and makes NADH
  • ∆𝑮°’ is positive (unfavourable)
  • generates NADH (only time in glycolysis) and BPG (first intermediate with a high phosphorylating)
  • better at transferring its phosphate to a donor than ATP) (it can directly generate ATP, next step
216
Q

describe step 7 of glycolysis

A
  • Substrate-level phosphorylation = Phosphoglycerate kinase
  • step where we take VPG and make 3PG and synthesizes ATP
217
Q

which two steps in glycolysis are thermodynamically coupled?

A

6/7
Step 6 is positive and Step 7 negative
- They are coupled and makes it negative, driving the
forward flux of glycolysis

218
Q

Describe step 9 of glycolysis

A
  • Enalase
  • turn 2PG into PEP
    PEP: second high energy intermediate (almost double hydrolysis of ATP)
219
Q

Describe step 10 of glycolysis

A
  • PEP makes pyruvate and ATP (pyruvate is used an an intermediate/co-factor as well)
220
Q

Overall points of glycolysis

A
  • Glucose (lots of energy stored)
  • Release energy until G6P
  • Irreversible
221
Q

what is the Pasteur effect

A
  • rate of fermentation decreases in the presence of oxygen
  • (ex: exposing anaerobic yeast to oxygen)
  • Oxygen decreased the rate of glucose utilization
  • regulation (oxygen causes yeast (non-living under oxygen condition) to stop metabolizing glucose))
222
Q

Name 2 major regulators for ATP and glycolysis

A
    1. Adenylate charge (ATP to ADP ratio)
    1. Fuel store (cell can sense how much fuel it has)
223
Q

how is hexokinase regulated?

A
  • Regulated by product inhibition
  • When G6P levels rise, it will feedback and inhibit
    the production of more G6P
224
Q

how is Phosphofructokinase regulated by adenylate charge?

A

How can it be a substrate and an inhibitor? (cell can sense how much energy it has)
- the active site has high affinity for ATP, ATP will bind the active site when levels are low
- Another site can bind to ATP, allosteric site, only when ATP levels are higher
-

225
Q

how is Phosphofructokinase regulated by F2, 6 BP?

A
  • F2, 6 BP is generated by PFK-2/FBPase-2 (PFK not the same as PFK-2)
  • Takes F6P and makes F2,6 BP
  • Not in glycolysis, but makes metabolic intermediate that
    inhibits PFK
226
Q

discuss how pyruvate kinase is regulated

A
  • Inhibited by high adenylate charge and high fuel stores
  • ATP is an inhibitor
  • Acetyl CoA is an inhibitor (made in mitochondria, so if acetyl CoA built up, which comes from fatty acids oxidation (sign that there’s enough and we can stop glycolysis)
  • Another way the cell senses how much fuel/charge they have
227
Q

what is PDC and what is its purpose

A
  • Pyruvate dehydrogenase complex
  • occurs in mitochondrion
  • makes acetyl CoA from pyruvate (from glycolysis)
  • pyruvate + NAD+ + CoA –> actely coA + NADH + CO2
228
Q

does PDC transport pyruvate into mitochondrion

A

NO, its already there.
- brought by mithocndrion pyruvate complex (MPC)

229
Q

how does pyruvate entering mitochondrion not mess up the gradient?

A

It enters with a symport which will bring in a protons to neutral things out (pyruvate is negative)

230
Q

Describe the steps of PDC and explain any co-factor needed

A

STep 1: E1 commits PDC to acetyl CoA formation by releasing Co2
step 2: regenerates TLL (co-factor for E1) so that it is not stuck
step 4-5: required to make acetyl coA AGAIN
- recycles e-
- generates acetyl coA (CAC) and NADH (oxidative phosphorylation)
step 5: NAD generates NADH, oxidizing FADH (E3)

231
Q

If FAD is oxidized and then reduced, how do we regenerate the enzyme?

A

NAD+

232
Q

What are the mechanistic advantages of multi-enzyme complexes?

A
  1. less distance for substrate between active sites (less need to maintain large pool of intermediates)
  2. coordinated control of reaction (which enzyme to shut off to shut the whole system down)
  3. metabolic intermediates are channelled between successive enzyme sites
    • protection of chemically versatile intermediates
    • side reaction are minimized
233
Q

Explain how PDC is regulated by allostery

A
  • product inhibition (accumulation of acetyl CoA and NADH will inhibit PDC)
  • Prevents useless consumption of pyruvate
  • Done by reversal: enzyme goes into reverse
234
Q

Explain how PDC is regulated by phosphorylation

A
  • covalent regulation (phosphorylation)
  • phosphorylate E1 when you want to shut it off
  • PDH kinase shuts it off
  • PDH phosphatase turns it back one (puts phosphate back)
    PDH kinase is allosterically regulated:
  • when levels of acetyl CoA, NADH and ATP are high, PDH kinase is shut off
  • when levels of pyruvate and ADP are high, PDH is turned on
235
Q

does CAC belong to aerobic or anaerobic metabolism?

A

NEITHER, it supports aerobic but never consumes O2 directly itself

236
Q

is CAC anabolic or catabolic? DESCRIBE each or both in either case

A

BOTH : its amphibolic
Anabolism:
-CAC intermediates are starting point of anabolic pathway
Ex: 1. Gluconeogenesis
2. Fatty acid synthesis
3. Amino acid synthesis
-Cataplerotic reactions (cata = emptying) deplete the CAC intermediates (syphon off)
Catabolism:
- Aerobic catabolism of carbohydrates/lipids/amino acids merge into the CAC
Ex: 1. Oxaloacetate (from pyruvate carboxylase) 2. Amino acid degradation
- Anaplerotic reactions (ana = filling up) replenish the depleted CAC intermediates

237
Q

Describe functions of CAC and what it produces

A
  • Produce reducing equivalence
    (NADH)
  • Produce intermediates for biosynthesis
  • harvest energy
238
Q

overall reaction of CAC

A

3 NAD+ + FAD + GDP + Pi + acetyl-CoA –>3 NADH + FADH2 + GTP + CoA + 2 CO2

239
Q

how can we make GTP from CAC

A

because CAC has an intermediate called succinyl-CoA ( similar to acetyl Coa) which is has a high energy bond being able to generate GTP directly

240
Q

State the net production of of CAC

A

3 NADH (2.5 atp/NADH) = 7.5 ATP
1 FADH2 )1.5ATP/FADHS2= 1.5 ATP
1 GTP = 1 ATP
TOTAL: 10 ATP (20 per glucose)

241
Q

what is the balanced sheet for the aerobic metabolism? (Cytosol, Mitochondria…)

A

Cytosol:
Glycolysis (from one glucose)
2 ATP = 2 ATP 2 NADH X 2.5 ATP/NADH = 5 ATP
Total 7 ATP

Mitochondria:
Pyruvate dehydrogenase (from 1 pyruvate) 1 NADH X 2.5 ATP/NADH = 2.5 ATP
Total 2.5 ATP

CAC (from 1 acetyl-CoA)
3 NADH X 2.5 ATP/NADH = 7.5 ATP 1 FADH2 X 1.5 ATP/FADH2 = 1.5 ATP
1 GTP X 1 ATP/GTP
Total = 1 ATP 10 ATP

Grand total:
Glycolysis (per glucose)
PDC (2.5 ATP x 2 pyruvate/glucose) =
CAC (10 ATP X 2 acetylCoA/glucose) = 20 ATP
TOTAL 32 ATP/glucose (per glucose)

242
Q

how do inhibitors are activator build up in CAC

A
  • inhibitors are metabolite that would build up if the cycle stops
  • activators (upstream) if they don’t get converted quick enough they build up
243
Q

CAC flux is responsive to:

A
  1. Energy state of the cell through allosteric activation (ADP/ATP/NADH)
  2. Redox state of the cell through the mitochondrial NADH/NAD ratio (things build up)
  3. Availability of energy rich compounds (acetyl-CoA, succinyl-CoA) that inhibit CAC Enzymes (CS and alpha-KGDH)
244
Q

whats the standard reduction potential?

A

how much a molecule wants to accept or donate e-

245
Q

is mitochondrion always dividing? is their outer membrane made up of critter?

A

YES! mitochondrion are constantly going through fission (breaking apart and mixing product)
- NO! inner part is highly invaginated (cristae)

246
Q

oxidative phosphorylation: where does it occur?

A

mitochondrion

247
Q

explain the steps of oxidative phosphorylation (complexes, ATP? e-? protons?…)

A
  1. Take oxidized e- from the nutrients
  2. Put e- onto reducing equivalence (NADH)
  3. NADH is oxidized to NAD
  4. e- that NADH was carrying pass through the
    yellow line to oxygen to make water
  5. As e- travels, conformation changes occur allowing protons to be pumped from the matrix into the inner membrane space (complex 1,3,4)
248
Q

which complex make up respiratory chain

A

1, 2, 3 and 4

249
Q

which complex does not pump protons? what does it do instead with the help of who?

A

complex 2
- succinate dehydrogenase
- FADH to FAD+

250
Q

how do e- get from complex 1 to 3? what about complex 3 to 4?

A

1-3: co-enzyme Q
- Succinate dehydrogenase has FADH and dumps e- on Co-Enzyme Q
1-4: co-enzyme C

251
Q

how many NADH, e- and protons are pumped in oxidative phosphorylation?

A

1 NADH = 2e- associated
2 e- that pass = (complex 1 = 4 protons, 3 = 4, 4 = 2)
(Total = 10 protons pumped)
1 oxygen atom consumed to water = 2 e- must past through = 10 protons pumped

252
Q

difference of ATP levels from NADH and FADH oxidation and why

A

NADH oxidation = 2.5 ATP
- FADH oxidation = 1.5 ATP
- because FADH doesn’t touch complex 1, goes to complex 2 first (doesn’t pump e-)

253
Q

what does pumping protons provide? explain further the gradient occurring

A
  • energy storing
  • electrochemical gradient= disequilibrium of high protons of intermembrane space compared to the matrix (regulated)
  • 30 million volts/meter = equivalent to lighting
254
Q

In the oxidative phosphorylation, we essentially go from NADH to O2 (big picture. describe which is acceptor/donator and standard reduction potential. Also give equation

A

NADH + H+ + 1/2O2 à NAD+ + H2O = -220kJ/mol
(220 is enough to produce ATP)
- NADH = -0.32 V = wants to give e- away
- O2 = + 0.82 = e- acceptor
- Difference of standard reduction potential between any 2 molecules and see how much energy was released

255
Q

discuss the evidence (6) backing up the hypothesis that ATP is synthesized from a high energy intermediate of the respiratory chain during oxidation.

A
  1. The respiratory chain can function in the absence of phosphate (ATP is not dependent on phosphate)
  2. The # moles of ATP generated through NADH oxidation was not an integer (suggesting intermediate steps required)
  3. An intact Inner mitochondrial membrane (IMM) is required for OXPHOS (generate ATP) ( this suggest that ATP synthesis must occur in mitochondrion and likely involves components of the respiratory chain
  4. Key electron transport proteins span the IMM
  5. Uncouplers such as 2,4-Dinitrophenol (DNP) inhibit
    ATP synthesis (coupling of ATP and e- transport is necessary for ATP production)
  6. Generating an ARTIFICIAL proton gradient permits ATP synthesis without electron transport (proves ATP synthesis is closely linked to proton movement across inner membrane)
256
Q

Why does the oxidative phosphorylation have so many redo reactions?

A
  • Bringing e- and oxygen to make water in 1 reaction would result in heat
  • Multiple steps are requires so the cell can harness a little
    energy for ATP
257
Q

explain how ATP synthase occurs across the membrane. Explain how it rotates. (describe strcuture)

A
  • ATP synthase is 13 proteins which rotate 360 to produce ATP
  • Rotates because as protons go down the channel they will push the F0 subunits (8 of them) causing C ring to spin
  • For every 360 turn, 8 protons go through
  • For every 360 turns, the ATP synthase makes 3 ATP
  • (8/3= 2.7 protons for every ATP molecule)
258
Q

describe the stoichiometry of OXPHOS and what the P/O ratio is

A
  • ratio: # ATP molecules for every oxygen atom consumed
  • P/O ratio through Complex I: 10/3.7 = ~2.5 (NADH)
  • P/O ratio through Complex II: 6/3.7 = ~1.5 (FADH)
259
Q

explain the discrepancy between # of protons coming out of ATP synthase vs how many we actually get.

A

2.7 - 3.7
ADP requires inorganic phosphate, but since it is negative and doesn’t wan tot disrupt gradient, symporter brings a proton to neutral it out
2.7 + 1 proton bought = 3.7 protons

260
Q

Name 5 eukaryotic models

A
  1. S. cerevisiae
  2. C. elegans
  3. D. melanogaster
  4. Danio rerio
  5. mus musculus
261
Q

Explain S. cerevsia (cellular type, generation time, haploid?diploid?, and its life cycle)

A
  • unicellular fungus
  • Generation time: 2-3 hours
  • Can exist as haploid or diploid
  • Can reproduce sexually or asexually
  • Can be frozen and revived
    LIFE:
  • can be haploid and make haploid offspring (alpha and a)
  • can be diploid and under starvation or temperature stress diploid yeast can go through meiosis and produce haploid cell
262
Q

Name advantages for haploid vs diploid cell

A

Advantage of haploid:
- only one copy of every gene in the cell, so knowing the effects of any gene is easy - gene can easily be mutated and see what effects there are on yeast
Advantage of diploid:
- understanding relationship between different genes

263
Q

Explain C. elegant (cellular type, generation time, haploid?diploid?, and its life cycle). Describe its freezing ability (what is it?)

A
  • Invertebrate animal, multicellular
  • Generation time: 3 days, 300 progeny
  • simple, translucent
  • Has invariant development: very single adult C. elegant is made up of the exact number of cells that develop in the exact same way
  • means we can trace fate of each cell (you know what it should look like)
  • sexes: male & hermaphrodite
  • Can be frozen and revived
    LIFE:
  • eggs are layer, forms larvae, matures to reproductive state and lays more eggs
    Freezing abilities: due to the Dauer stage:
    1. If larvae are exposed to difficult life conditions they can go into this state
    1. State of hibernation (freeze them)
    1. Revive them months later and they can reproduce
264
Q

Explain D. melanogaster (fruit-fly)(cellular type, generation time, haploid?diploid?, and its life cycle)

A
  • Invertebrate animal, multicellular (more similar to humans: skeletal and body plan)
  • Generation time: 10 days, 100 progeny
  • More complex than C. elegans
  • Share 75% of human disease-causing gene
265
Q

Explain zebrafish (danio rerio)(cellular type, generation time, haploid?diploid?, and its life cycle)

A
  • Vertebrate animal, multicellular
  • Generation time: 2-3 months, 200 eggs
  • Optically translucent embryos & larvae
  • Relatively simple & inexpensive to maintain
  • Easily treated with small molecules for drug & toxicity screens
  • Closer to some human concept than mice (ex: melanoma)
266
Q

What is the difference between - Axolotl (cute amphibian) and Planaria (flat worms)

A
  • Axolotl (cute amphibian): can regenerate limbs (to a normally functional limb)
  • Planaria (flat worms): can regenerate everything (its entire body)
266
Q

Explain mus musculus (mice) (cellular type, generation time, haploid?diploid?, and its life cycle)

A
  • Vertebrate mammal
  • Generation time: 3 months, 2-12 pups
  • Small, easy to house (for mammals)
  • Mice are NOT always perfect models for humans !!
267
Q

Forward vs reverse genetics

A

forward: phenotype to genotype
reverse: genotype to phenotype

268
Q

how do we forward genetic

A
  1. perturb gene
  2. see how gene reacts (phenotype)
  3. Try to see what gene caused it
269
Q

what are Temperature sensitive mutations

A

In restrictive temperature (warmer), it affects the folding of the protein, or the product protein isn’t functional

270
Q

explain the process of Replica plating to isolate temperature sensitive mutants

A
  • mutated yeast on a plate
  • replicate plate and stick to velveteen (sticky)
  • stick to many plates and grow each colony in different temperatures
    Low temperature: should have original colonies
  • High temperature: colonies that cannot grow should be present here (temperature sensitive mutants!)
271
Q

explain the 3 cdc mutants having to do with cell cycle defective mutants

A
  1. cdc1: cells who already formed bud were able to divide, but new ones were frozen (result= cdc1 involved inc ell wall formation)
    - frozen at G1 phase
  2. cdc2: cells formed buds, but when they tried to replicate they got frozen (S phase= DNA replication) (result= cdc2 involved in DNA polymerase)
  3. cdc3: defect in late stages of mitosis (replication occurs but daughters cannot separate) (something wrong with septin = cleavage)
272
Q

discuss experiment done with cdc 2 and 6 (complementation test) and what was found.

A
  • we wanted to know if mutants that are frozen at the same stage are from same gene or different genes affecting sam process
    • 2 yeast strain: cdc 2, 6 with similar phenotype
      Mate the 2 strains:
    1. If offspring is fine = different genes (1 mutant copy from cdc 2 and wild type from cdc 6)
    1. If offspring is NOT fine = same gene (mutant copy from both)
273
Q

describe experiment done with yeast where we tested to see if mutants rescues phenotype.

A
  • take all yeast DNA and cut it and clone it to plasmid
  • introduce to bacteria (way to store it)
  • introduce different Yeats into Yeats and see which plasmid rescue mutant
  • sequence plasmids that recuse and identify yeast gene
274
Q

describe experiment done with humans equivalent to yeast where we tested to see if mutants rescues phenotype.

A
  • CDNA library
  • introduce cDNA into Yeats and see which recuse mutant
275
Q

discuss the equivalent of cdc28 and cdc2 in humans how we found it.

A

cdc2 (S. pombe) = cdc28 (S. cerevisiae) = Cdk1 (human)
HOW:
- make human cDNA
- transformed plamids to temperature sensitive mutants
- plate them at high temps = Occasional colonies survived: plasmids being able to rescue the yeast
- Found that Cdk1 in humans is HIGHLY conserved (from humans to yeast) and you can take it into Yeats and recuse genes

276
Q

discuss discovery of ced-1/ced-3

A

ced-1 : required for engulfment (not cell death)
- you can see dying course on it can’t be absorbed
- did a screening test with ced-1 background and found ced-3 (mutant) which made cell death go away
- ced-3 wild type is apart of cell death (normally)

277
Q

how many cell form/make it to adulthood in c. elegans

A
  • ## normla c. elegans have 1090 cells during embryogenesis, but only 959 make it to adulthood = 131 die
278
Q

describe Egl-1 and its mutant problems. describe the finding of red-4

A

Egl-1 mutants have too much cell death which leads to their inability to lay eggs
- they need HSN (neuron required to lay egg)
- too much cell death kills HSN)
Ced-4: found that ced-4 mutant prevents excessive cell death, allowing HSN to survive
- tells us that ced-4 is required in cell death

279
Q

describe ced-9 mutants and what happens with cell death

A
  • when ced-9 is mutated (absent/off) it kills every cell in embryo
  • when ced-9 is overexposes, it suppressed cell death (none happening)
  • similar to BCL2 in humans (lymphoma related)
280
Q

describe how Human Bcl-2 can modulate cell death in C. elegans (experiment)

A
  • take wild type worm and put little pieces of DNA with heat inducible promoter
  • # of cell were normal before and after heat shock
    1. do same thing but with ced-9
  • # of cell before heat shock = normal
  • # of cell after heat shock = too many! (ced-9 was overexpressed and didnt allow cell death to occur)
  • same thing happens in bcl-2: it repressed cell death when overexpressed
281
Q

how can cell death occur

A
  • cell can get extrinsic stimuli from neighboring cell
  • Cell can have internal problem (DNA damage, not enough nutrients)
282
Q

Explain BLC-2, BAK and BAX

A

BLC-2 inhibits BAX and BAK
- 1. BCL-2 releases BAX and BAK (becomes active)
2. form a pore that permeabilizes the mitochondrial membrane and allows molecules to leak out
3. Mitochondrial cytochrome c is releases, which activates Apaf1
4. Apaf1 activates caspase 9 (proteases that chew things up)
- 5. Caspase 9 cleaves and activates caspase 3 (causes irreversible death)

283
Q

Compare all the ced-… with human genes

A
  • C. elegans ced-9 = human BCL-2 (loss of ced-9 causes more death)
  • C. elegans ced-4 = human Apaf1 (loss of ced-4 causes less death) (can’t sense the pores)
  • C. elegans ced-3 = human Caspase3 (loss of ced-3 causes less death) (can’t chew things up)
  • Egl-1 inhibits ced-9 and the original mutation was a gain of function! (more egl-1 activity causes more death)
284
Q

what is a heterochronic mutants. give 2 example

A

have defects in development timing (tree of linages gets perturbed)
lin-14
let-7

285
Q

explain tree lineage of different lin-14 depending on what they are missing. also what do lin-14 do? develop too quick or too slow?

A
  • Lin-14 mutants develop too quickly (loss of function) or too slowly (gain of function)
286
Q

explain lin-14 protein levels and lineage tree… and lin-4

A
  • wildtype: proteins levels only present at L1 stage than goes away
  • lin-14 loss of function: no proteins (skipped L1 stages)
  • lin-14 gain of function: too much proteins for too long (keep repeating 1 stage protein levels)
    Lin-4: looks like lin-14 gain of function
  • Lin-4 = repressor for lin-14
287
Q

does lin-4 code for a protein?

A

NO

288
Q

what did northern blotting lin-4 tell us about its size and component

A
  • extract RNA, run of gel, probe and see size
    1. Lin-4S (21bp, linear): Short RNA not present in mutant
      1. Lin-4L (60bp, hairpin): Long RNA found in both mutant and wildtype
  • found by overexpressing Lin-4
289
Q

what does lin-14 having two small RNAs mean for complementation to Lin-14

A

Lin-4 RNAs are complementary to lin-14 mRNA (3’ UTR)
- Short Lin-4 RNA are binding to the RNA of Lin-14 (somehow regulating lin-14 levels)

290
Q

discuss let-7 and what type of mutation it is

A
  • heterochonic mutation
  • did the same thing as lin-4 and found a small fragment of 21bp
  • found it cpmpelmeneted to lin-41 (another development thing) and to Lin-14
291
Q

describe miRNA precessing and activity

A
  • transfixed as 70bp and then cleaved by drosha (form stem loop 70bp)
  • gets exported to cytoplasm
  • gets cleaved by dicer into 21bp small fragment
  • gets incorporated by argonaut to RNA induced silencing complex
  • Binds 3’ UTR (not coding sequence) and downregulates the RNA
292
Q

how can miRNA regulate larger target

A
  1. cleave making translating impossible
  2. bind to them and unable them to translate
  3. remove poly-A tail and 5’ cap
293
Q

are miRNA perfectly complementary to target?

A

NO
- have complementary to the 3’ and 5’ ends
- 1 miRNA can bind to multiple targets

294
Q

what happens in miRNA for vertebras

A

tend to be “gentle” regulatory mechanism important for fine- tuning gene expression levels

295
Q

discuss SEV mutations in Drosophila

A
  • their (compound eyes) eyes contain repeating arrays of photoreceptor (ommatidium)
  • Sev mutants: cannot form R7 (important for UV and colour)
    raise indirect immunofluorent against Sev and notice that it Is located at PM (TM tyrosine kinase = receptor)
296
Q

Discuss Sevelessn (boss) mutation in drosophila

A
  • cannot form R7
  • located on R8
  • binds and activated R7
  • DOWNSTREAM SIGNALING PATHWAY
  • ligand
  • required for formation of R7 but is not expressed in r& but aroundd it
  • meaning that Boss plays a non-autonomous role in R7
297
Q

Explain Sev (LOF), (GOF), and (SOS)

A

Sev (GOF): Gain of function Sev mutation - Receptor is hyperactivated (doesn’t need ligand to be activated)
- Extra R7 cells

Sos (LOF): Son of Sev (loss of function)
- When combined with Sev (GOF) it will
bring things back to normal (single R7 cell)
- Heterozygous mutant (one normal/one loss of function mutation= enough to suppress GOF but not enough to kill fly)

298
Q

Explain the mechanism of the downstream components of the seven less RTK pathway

A
  1. ligand binds receptor causing clustering of Sev and they phosphorylate each other
  2. phosphorylation recuits scaffold protein (Drk)
  3. Drk recruits Ras-GEF (SOS)
  4. Sos facilities the exchange of GDP to GTP
  5. Results in downstream signals
    important for R7 specification
299
Q

Name both classes of Mobile elements. Which one is more helpful for genetic screening?

A
  1. cut/paste: Individual transposon DNA region jumps around in the genome (more helpful for genetic screening)
  2. copy paste:
    - Elements that integrate into the genome of host and transcribe into RNA
    - RNA is reverse transcribed into another DNA that gets integrates into
    another place in the genome
300
Q

What is the difference between gene trap and enhancer trap?

A

gene trap: interrupts a gene, causing loss of function (studying that loss to see where the gene is expressed by inserting reporter)
enhancer trap: Used to identify enhancers that promote gene expression in a specific cell type
- Inserted DNA does NOT need to land inside a gene (it has its own promoter)

301
Q

Explain P-element mechanism and which type of class of mobile element it is.

A
  • cut/paste: transposon encodes transposase (enzyme helping jumping DNA re-integrate)
    • Took out the transposase and put it on a separate plasmid
  • Took transposon with repeated sequences on each side and inserted elements (reporter = GF, LacZ = to see where target gene is expressed AND selection gene (ex: genes that affect eye color from red to white)
    • Take modified transposon + transposase and inject into a fly germline
  • Fly lays eggs and has offspring
302
Q

how do the first days of mammalian development go

A

ygote and cleavage stage embryo DON’T rely on RNA and protein that they made themselves
-Rely on maternally deposited RNA (made in female germline before hand) Embryo goes through Zygotic/Embryotic genome activation (ZGA / EGA)
- degradation of maternally
deposited RNA and zygotic genome becomes active (makes its own RNA)
- Up to this point: all cells in embryo are totipotent (all the same/all have the capacity to generate any cell type in the future organism)
- More flexible (taking out cells = organism will survive because other cells with compensate)
Embryo goes through Compaction: Increased of adhesions (cells stick to another)
- important for cavitation step (increased adhesions prevents leakage) Cavitation step: Pumps water in, generates fluid cavity important for morphogenesis
- forms Blastocoel cavity (embryo is now a Blastocyst)

303
Q

whats first lineage decision: (cells stop being totipotent (first round of restriction)

A
  1. Formation of cells outside the embryo form Trophectoderm lineage 2. Formation of cell inside the embryo form Inner cell mass lineage
    - Inner cell mass goes through another round of restriction: 1. forms Epiblast
  2. forms Primitive endoderm
    - Blastocyst (with 3 lineages) implants into the uterine wall and undergoes post- implantation development (pre-implantation dev. before diffusing through wall)
304
Q

Cell position guides early differentiation

A
  • Cells of trophectoderm will make the cells of the placenta
  • Cells of Inner cell mass will make the cells that make up your entire body
305
Q

How do the cells know they’re on the outside/ inside and what they should make?

A
  1. Inner cells are surrounded on all sides by other cells
  2. Outer cells are touching inside cells BUT also have an interphase with no contact to any cells (contact with fluid from the fallopian tubes)
    This is an important cue that triggers changes in a downstream pathway: hippo signalling pathways
    - In inside cells: there’s a transcription factor YAP that gets phosphorylated by LATS1/2 (kinases)
    - YAP cannot get into the nucleus, so can’t activate important genes for
    trophectoderm differentiation
    - In outside cells: F-actin accumulates
    on the interphase with no cell contact - F actin: Binds and sequesters
    LAST1/2 (pulled away from cytoplasm) making them unable to phosphorylate YAP
    - YAP can then move into the nucleus
    and can turn on genes important for trophectoderm differentiation
306
Q

when does epiblast go under gastrulation

A

After blastocyst implant in urinary wall: undergoes gastrulation

307
Q

explain gastrulation

A
  • Epiblast cells have a very broad potency (the potential to become/make any cell)
  • Gastrulation is when that potency is further restricted Schematic:
    • -
  • Primitive streak: site of gastrulation
    Single layer of epiblast cells: over time cells start to ingress (enter) and move through the primitive streak and migrate down
    3 main germ layers:
  • Initially: cells that migrate down form 1st layer (endoderm)
  • Over time: cells that migrate down forma 2nd layer (mesoderm) - Cells that do NOT migrate: turn into ectoderm
308
Q

ecto, meso and endo

A
  • Ectoderm: skin, neuron, melanocyte
  • Mesoderm: bone, muscles, blood
  • Endoderm: gut, lung cells
309
Q

example of anterior - posterior mutation

A
  • Mutation of Ultrabithorax (Ubx) causes homeotic transformation
  • Loss of Ubx: Duplication of wing segment and loss of haltere
    segment
  • Gain of Ubx: Opposite: no wings and duplication of haltere
    segment
  • Ubx is expressed in a specific region that would go on to make
    the haltere segment
310
Q

how can we visualize a posterior-anterior segment

A

Fluorescence in situ hybridization: take embryo and make RNA probe against sequence you’re interested in
- probe (with a fluorescence for visualisation) will bind to the sequence

311
Q

what is HOX

A

All genes stained in previous schematic are part of
HOX cluster
- HOX: evolutionary conserved group of genes
- series of transcription factors that are essential to the Anterior- Posterior Patterning
- Each gene is expressed in a specific domain of the fly embryo important for the fate to a specific part of the body (important to know which ay is the head and which way is the bottom)
- For the most part, they are arranged in the same order on the chromosome and on the body (see picture above)

312
Q

how do mice use AP axis

A

Mice use the same genetic strategy to pattern the A-P axis
- You can overexpress GOF or LOF of Hox and see same effects than in flies
- HoxA10: important for the patterning of lumbar vertebrae part of the body

  1. Overexpressing HoxA10 in all of the vertebrae: all the vertebrae become lumbar (no more thoracic)
  2. Loss of HoxA10: Loss of lumbar vertebrae and extension of thoracic vertebrae
313
Q

whats planar cell polarity explain

A

Hair follicles is a good way for cells to know which way is tail/head - All dog hairs go in the same direction
- How do hairs know which directions to go into? Planar Cell polarity pathway
- Pathway that transduces global A-P patterns to interspatial information at the level of individual cells
- Does this by: segregating different components to Anterior and Posterior of each cell

314
Q

describe neutral tube in CNS

A
  • Ectoderm forms the nervous system (from the neural tube)
  • A-P pattern is important for neural tube formation (which way to go)
  • Dorsal – Ventral pattern is also important: - Dorsal side of spinal cord: sensory

Inputs (pain)
- Ventral side: motor neurons (connect
to muscles and react to touching hot surfaces)

315
Q

explain morphogens

A

-
Diffusible signals that exert graded effects
1. Cell act as a source of morphogens (secrete smtg out into the environment) 2. That morphogens diffuses in every direction
3. Cells further away get less exposure to the morphogen
4. Different levels of morphogens are translated into different gene expressions program
- High levels will take a different fate than the low level ones Common Morphogens: Shh, TG𝛽, Fgf, Wnt

316
Q

describe morphogens in neural tube

A
  1. BMP (Dorsal)
  2. Shh (Ventral)
    - Depending on where the cell sits on the axis, it will get different combinations of signals
    Schematic (bottom):
    - Transcription factors on Dorsal or ventral side
    - Combination of different transcription factor
    expression allows neural tube to form different neural precursors (which will form different types of neurons in specific places in spinal cord)
317
Q

Signals are endlessly used & re-used in
development

A
  1. Combinatorial signaling
    - Cell in embryo is exposed to different sources, signals resulting in different combination of signals
  2. Cellular memory
    - Cells in embryo are exposes to different signals
    - Their differences are able to respond to the same signal in a different way
318
Q

How do we make an organism get bigger?

A
    1. Cell growth: cell # don’t change, but they grow overtime
    1. Cell division: make more cells
    1. Cell death: getting rid of cell (bad ones), prevents you from being smaller
      Schematic (left):
      -Common pathway that regulates these processes (specifically cell death/division):
      -YAP moves into nucleus (under specifics rules)
  • at later stages, it relates with proliferation
  • mature organism: overexpression of YAP = more growth/proliferation

ANAT 212 (2 decks)

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