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Molecular Biology L-211 > Exam 5: Lecture 7 > Flashcards

Exam 5: Lecture 7 Flashcards

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1
Q

Hippo Pathway-General

A
  • Tumor suppressor pathway used to control growth of tissues and organs
  • Entire pathway has been elucidated in both flies and mammals
  • Nearly all components are conserved across 500 million years of evolution
  • tasked with repressing activity of Yki homolog (YAP)
  • represents universal mechanism for controlling growth
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2
Q

Hippo Pathway-First Identified and Associations

A
  • via efforts to understand how a tissue or organ knows when to stop growing
  • since then closely associated with tumor formation and cancer
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3
Q

Mutations in Hippo Pathway

A
  • Fat4 and NF2 are underlying causes of breast cancer and schwannomas
  • main target of this pathway is Yki TF and is expressed at higher than normal levels in several breast, colorectal, and liver cancers
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4
Q

Founding Member of Hippo Tumor Suppressor Pathway (Shar-pei)

A
  • documented effects of removal of this gene from entire head and retina
  • wild type control animals have flat head cuticle surface
  • mutant tissue over proliferates making undulating folds of head capsule tissue
  • resembles shar-pei dog so that’s where it gets it’s name
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5
Q

Removal of Shar-pei

A
  • ability to suppress cell proliferation is not limited to head and retina
  • remove shar-pei within clone of cells within torax leads to tumor formation
  • loss of shar-pei throughout haltere leads to significant increase in size
  • in every tissue examined, shar-pei controls organ size throughout all developing Drosophila tissues
  • same shown for mammalian Hippo pathway
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6
Q

Why tissues appear larger: 1

A

-number of cells in wild type and mutant tissue can be the same, but the cells can be bigger in the mutant

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7
Q

Why tissues appear larger: 2

A

-size of wild type and mutant cells can be the same but the number of these cells can be significantly higher in mutant tissue

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8
Q

Fly Ommatidium

A
  • each ommatidium separated from neighbors by single cell
  • in shar-pei mutant there are more cells between ommatidia
  • results indicate Hippo pathway is a true tumor suppressor pathway and that its role in development is to suppress cellular proliferation
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9
Q

Mutant Clones

A
  • there are tricks one can employ to generate patches of cells that will be mutant for both copies of a single gene (-/-)
  • when mutant clones are generated, twin clone that is wild type for both copies of gene of interest also generated
  • growth features of two clones can be analyzed against each other
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10
Q

Mutated gene not involved in growth control

A

-size of mutant clone will be same as wild type twin spot

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11
Q

Mutated gene required for cell to grow

A

-mutant clone will be smaller than wild type twin spot

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12
Q

Mutated gene is tumor suppressor

A

-mutant clone will be larger than wild type twin spot

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13
Q

Drosophila Eye and Tumor Suppressors

A
  • w/t tissue marked with red pigment and mutant tissue lacks pigment
  • control: approximately equal amounts of red and white tissue
  • experimental: mutant tissue completely taken over entire retina
  • the gene that is mutant in this example is member of Hippo suppressor pathway
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14
Q

Why create mutant clones instead of looking at entire animals?

A
  • tumor suppressor genes usually expressed in all cells of developing organism
  • mutation that removes tumor suppressor gene from entire animal will die early in development due to tumor formation
  • generation of mutant clones in eye allow for rest of animal to develop normally
  • since eye is dispensable organ, fly containing retinal clones that are mutant for a tumor suppressor will survive to be analyzed
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15
Q

Hippo Pathway Componets

A
  • isolation of Hippo pathway components have been achieved due to variety of genetic and biochemical screens
  • with one exception, loss of any member of Hippo pathway leads to phenotypes seen in shar-pei mutants
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16
Q

Removal of hippo gene

A
  • leads to over-proliferation, tissue undulations, and increase in overall organ size
  • additional defects in planar cell polarity, cell junction integrity, and cell migration seen in some pathway members associated with the membrane
17
Q

Yorkie-inhibition

A
  • entire cytoplasmic hippo pathway focused on inhibiting activity of Yki TF
  • it encodes transcriptional co-activator that cannot bind to DNA on its own but interacts with DNA BP scalloped
  • Sd-Yki composite TF bind to enhancers of genes involved in promoting cell proliferation and tissue growth
  • Hippo pathway regulates Yki so tissues and organs stop growing when appropriate size has been achieved.
18
Q

Yorkie

A
  • member of Hippo pathway isolated in screens looking for growth regulators
  • however, mutant clones grew poorly when compared with w/t tissue
  • gene called Yorkie
19
Q

Yki removed from developing head and retina

A

-both tissues considerably smaller that w/t

20
Q

Yki expressed at higher than normal levels in developing wing disc

A

-tissue maintains overall shape but is considerably larger than w/t counterpart

21
Q

Indications of Yki removal/expression

A

-Yki promotes growth and Hippo pathway’s role in development is negatively modulated by activity of Yki

22
Q

Yki and Cell Proliferation

A
  • generate clones of w/t tissue these cells grow happily in wing disc
  • overexpress Yki clones are bigger
  • remove Yki then clones hardly grow at all
  • support idea that Yki promotes cell proliferation
23
Q

Bantam miRNA

A
  • one of the targets of Yki-Sd complex
  • remove bantam miRNA cells grow poorly
  • now remove bantam from clone that is overexpressing Yki, clones grow more poorly than if bantam is present
  • overexpress bantam miRNA in cells lacking yki, clones grow much better than clones that just lack yki
24
Q

Results of bantam/Yki experiments

A
  • suggest bantam is genetic target of Yki-Sd complex
  • idea that in cells that are growing Yki-Sd activates expression of bantam which in turn will bind to 3’ UTR of a set of target mRNAs
  • resulting degradation of target mRNA or the blockage of translation of that mRNA results in enhanced growth
25
Q

hid and 3’ UTR

A
  • miRNAs bind to 3’ UTR of mRNAs and stimulate degradation or block translation
  • search for putative targets identified head involution defective (hid) mRNA is putative target
  • within 3’ UTR of hid are five potential binding sites for bantam miRNA
26
Q

miRNA sensor assay

A
  • determine if particular 3’ UTR is true target of any given miRNA
  • fused 3’ UTR of hid to coding sequences of GFP
  • w/t wing disc mRNA translated normally and disc glows
  • Using UAS-GAL4 system forcibly expressed bantam miRNA along A/P axis
  • level of GFP expression dropped along A/P axis
  • indicates bantam miRNA was able to bind to 3’ UTR and block production of GFp
27
Q

Hid and Cell Death

A
  • member of programmed cell death pathway
  • in response to apoptotic stimuli hid and other proteins initiate cascade of events leading to death of cell
  • different versions of pathway
  • in fly embryo significant amount of programmed cell death
  • caused by overproduction of cells during early stages of development
  • excess cells must be pruned away prior to hatching of embryo into larva
  • in mutants that lack hid gene all cell death is blocked
  • overexpress hid within developing eye, high levels of cell death induced and only tiny eye is produced
28
Q

Cells that are growing

A
  • Yki-Sd complex activates expression of bantam miRNA which in turn binds to hid mRNA and blocks production of Hid protein
  • without this key cell death inducer apoptosis is blocked and cells can proliferate
  • when it’s time for cells to stop growing, Hippo pathway blocks activity of Yki-Sd
  • results in loss of bantam expression
  • without bantam miRNA hid mRNA can be translated
  • Hid protein can then induce cell death and prevent tissue or organ from growing out of control
29
Q

Hippo Pathway and Cancer

A
  • several tumors and cancers in humans are associated with loss of several different Hippo pathway elements
  • oss of Hippo signaling in mouse liver leads to dramatic increase in size of organ
30
Q

Mst1 and Mst2

A
  • homologs of Drosophila Hippo protein
  • loss of either individually has no effect on organ size
  • both genes must be simultaneously eliminated in order to see an effect on cell proliferation
  • Mst1 and Mst2 are products of duplication event that occurred after split in insects and vertebrates.
  • since individual mutations have no phenotype it is thought that the genes are redundant in function

Molecular Biology L-211 (38 decks)

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