1. daf-2 mutants, when down-regulated in the L2 stage, cause constitutive dauer formation (Daf: abnormal Dauer-formation) and arrest in the L3d-dauer stage. However, you have also heard that down-regulation of daf-2 during adulthood is sufficient to increase lifespan of C. elegans. This causes an experimental problem for aging researcher studying daf-2. How can we dissect dauer formation and lifespan extension experimentally?
a. Think of experimental ways to study (adult) lifespan in C. elegans without letting worms go to dauer.
b. How can you prove that the daf-2 (mutant) induced lifespan extension depends on daf-16?
a)
Conditional knockout of daf-2 e.g temperature sensitive - growing worms on specific T till they eare young adults - avoids dauer formation and after that shift temperature for KO to study lifespan extension
RNAi Transgene & inducible promotors (e.g. tet) - problematic: might not completely eliminate daf-2 expression (partial effects),off-target effects, impacting other genes related to insulin signaling -> might affect organism significantly
Inhibitors: chemical inhibitors that specifically inhibit daf-2 activity only in adult worms no dauer formation focus on lifespan extension - possible systemic effects
b) Double mutant daf-2 & daf-16 -> does this affect longevity effect ?
If yes indicates that daf-16 is required for daf-2 mediated lifespan extension
2. What is the role of PIP2 and PIP3 in insulin/IGF signaling?
PIP2 and PIP3 function as key signaling molecules that regulate the activation and downstream signaling of AKT in the insulin/IGF pathway, influencing cellular processes such as metabolism, growth, and survival.
PIP2 (phosphatidylinositol 4,5-bisphosphate) & PIP3 (phosphatidylinositol 3,4,5-trisphosphate) are docking lipids anchored in the membrane and act as signaling molecules in the Insulin/IGF pathway.
PI3K converts PIP2 to PIP3 by phophorylation and PTEN phosphatase reverses this
PIP3 activates PDK1 (3-phosphoinositide - dependent protein kinase 1) -> PDK1 phosphorylates AKT
AKT can bind PIP3 -> Initiation of downstream phosphorylation cascade
3. PTEN is generally knows as a tumor suppressor gene, its C. elegans homologue is daf-18. What is the (biochemical) function of PTEN, what is its substrate?
What are the consequences of losing PTEN or daf-18 activity?
Would you expect an increased or a decreased risk of tumorigenesis?
Provide arguments for your conclusion, and try to interprete the loss of PTEN mechanistically (what are the consequences in the signaling pathway?).
PTEN (Phosphatase and Tensin Homolog) functions as lipid phosphatase -> dephosphorylates its substrate phosphatidylinositol 3,4,5-trisphosphate (PIP3) to phosphatidylinositol 4,5-bisphosphate (PIP2).
Function: negatively regulate the PI3K/AKT signaling pathway by reducing PIP3 levels
Consequences of Loss of PTEN or daf-18 Activity: increased PIP3 levels, -> hyperactivation of the pathway through consitutively active PDK1
Expected Risk of Tumorigenesis: increased risk of tumorigenesis: hyperactivation of the pathway promotes cell growth, survival, and proliferation, while inhibiting apoptosis
constitutively active pathway through elevated PIP3 levels - prolonged AKT activation - phosphorylation subsequent activation of downstream targets -> cell survival & growth promoting pathways
4. Which genetic mutations (mutations in which gene) in C. elegans would you consider to be equivalent to the defects observed in Type I vs. Type II Diabetes?
Discuss this !
Type I Diabetes: Results from autoimmune destruction of insulin-producing beta cells (islet cells) - always require insulin injections
Genes involved in Insulin production
Type II Diabetes: Results from insulin resistance, where target tissues become less responsive to insulin despite its production - often obesity related
Genes involved in insulin reception or downstream pathway
Insulin/IGF Receptor ( DAF-2)
IRS1 (insulin receptor substrate that is recruited to the membrane via the lipid binding sites PIP3 -> activating form
PI3K (AGE-1)
PDK1 -> AKT / SGK-1
5. pdk-1 mutants in C. elegans result in the loss of sgk-1 expression in the intestine. This was interpreted as SGK-1 being downstream (and a substrate of) PDK-1 and, based on other experiments not shown here, upstream of DAF-16. We did not discuss this during the lecture, but one slide shows some novel results, according to which sgk-1 may also be a transcriptional target of DAF-16 (see diagram on the next page). Think of experiments that could distinguish between both possibilities.
??? Discuss
Genetic Epistasis Analysis:
Generate double mutants with pdk-1 and daf-16 mutations
Assess SGK-1 expression levels in these mutants compared to single mutants.
SGK-1 expression is restored or altered differently in the double mutant compared to the pdk-1 single mutant, it suggests that DAF-16 acts upstream or parallel to SGK-1 regulation.
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