Lab 4

Question 1

Let 363

Answer 1

A kinase that, when cell has high energy, blocks ampk from interacting with autophagy regulators to keep them OFF so there’s no autophagy

Question 2

Ampk

Answer 2

Under low energy (ATP), AMPK (AMP-activated kinase) is activated; AMPK phosphorylates proteins that trigger autophagy initiation

Question 3

ROS

Answer 3

Reactive oxygen species. at low levels, activates food signaling pathways. at high levels, can cause cell damage

Question 4

ATFS1

Answer 4

Transcription factor that activates transcription of genes that make proteins used for proteostasis

Question 5

UPRmt

Answer 5

mitochondrial unfolded protein response.
Definition: the transcriptional and cellular response activated upon mitochondrial dysfunction

Question 6

UPRmt causes

Answer 6

● accumulation of misfolded proteins in the mito matrix
● perturbations (for ex, knocking down gemes) to Oxphos activity (oxidation phosphoryl. the complexes we’re studying)
● disrupting mitochondrial’s ability to import
○ (due to lack of ATP or electrochemical gradient) bc to transport stuff, mito uses atp
● mitochondrial DNA defects: can lead to protein defects (bc dna makes proteins) and defective mito

Question 7

possible hypothesis for exp 3

Answer 7

Mild mitochondrial disruption may promote stress resistance through upregulation of the mitochondrial unfolded protein response which alters signaling, cellular response and gene expression to help cell cope w defects

Question 8

what are the 4 worm conditions

Answer 8

wt (no knockdown), wt (goi knockdown), q35 (no knockdown), q35 (goi knockdown)

Question 9

what should you be doing when writing research proposal

Answer 9

convince the reader your set of experiements are worthwhile. (you should be convincing an agency that they should give you $ to perform experiment.) discuss where field is at, what data you’ve found, and where you’re going

Question 10

WHY does polyp aggregate hurt cell health? Why would expression of an aggregation prone protein be a problem? Wouldn’t the cell just clear it away?

Answer 10

This is an active area of research! Some hypotheses:
★ Misfolded proteins trigger continual stress response
★ Titrate away chaperones from normal cell functions
★ Aggregates accumulate other proteins with them, removing them
from their normal function
★ Proteostasis network and autophagy function declines during aging

Question 11

Positive control

Answer 11

We know what’s expected. For example: to see how much rfp is made, heat shock is our positive control because we know heat shock activates rfp expression

Question 12

hsf 1

Answer 12

transcription factor

Question 13

explain process of hsf 1, starting when chaperones are bound to them

Answer 13

chaperones bind to hsf 1 to inactivate them. as more misfolded proteins enter cell, chaperones leave hsf1 and hsf1 then forms a trimer. this trimer can bind to dna sequence in the promoter region of a gene (the heatshock responsive genes that make heatshock responsive proteins. thus the genes will be turned on because of the misfolded proteins). many heatshock responsive genes are chaperones- so when chaperones leave hsf1, this frees hsf1 to go form trimers, bind to promoters of chaperones, and make more chaperones/proteostasis proteins.

Question 14

when we disrupt mitochondria, we’re asking…

Answer 14

does this lead to enhancement of expression from heat shock responsive genes?