1 / 10

The Unfolded Protein Response in C. Elegans

The Unfolded Protein Response in C. Elegans. Biology 314, Advanced Cell Biology, Spring 2004. Characterization of a Mutation in the Unfolded Protein Response Signal Pathway of the Nematode Worm C. elegans. Jillian Anibaldi, Chris Binner, Jo’Vonna Grayson & Artemio Linares

beryl
Download Presentation

The Unfolded Protein Response in C. Elegans

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. The Unfolded Protein Response in C. Elegans Biology 314, Advanced Cell Biology, Spring 2004

  2. Characterization of a Mutation in the Unfolded Protein Response Signal Pathway of the Nematode Worm C. elegans Jillian Anibaldi, Chris Binner, Jo’Vonna Grayson & Artemio Linares BIO 314, Advanced Cell Biology The unfolded protein response (UPR) is a transcriptional and translational intracellular signaling pathway activated by the accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER). Protein folding in the endoplasmic reticulum is sensitive to agents that interfere with protein glycosylation or the formation of disulfide bonds (Tunicamycin or DTT). A strain of mutant C.elegans, SJ17, has been identified as having a flaw in the UPR. We have further characterized the mutant strain by: (i) assaying the mutants for expression of hsp-4 gene, a characteristic of the UPR. We have shown that the mutants are incapable of inducing hsp-4 when stressed with tunicamysin, (ii) The mutants,treated with DTT and Tunicamycin, exhibit slow growth and do not progress beyond the L2 larval stage. The observed deficiency in UPR, could lie in ire-1 since this gene codes for the endonuclease that signals ER stress by splicing (and activating) the mRNA for Xbp-1. Xbp-1 is a transcription factor, which activates the hsp-4 and other genes, required for the UPR. We are currently attempting to determine if the xbp-1 mRNA in the mutant strain is spliced in response to stress. If xbp-1 is spliced, then ire-1 is not the gene in which the mutation lies. The mutation, then, might be in the xbp-1 gene or other distal elements of the pathway.

  3. Introduction • The Endoplasmic Reticulum (ER) is the compartment in cells where membrane and secreted proteins are folded and glycosylated. • When proteins fail to fold properly in the ER they can potentially aggregate and eventually kill the cell. • Cells prevent aggregation by synthesizing molecular chaperone proteins which assist proper folding. • When the ER is stressed by agents which interfere with glycosylation or folding, the cell responds with the synthesis of additional chaperones. This response is called the UPR (See next panel for description). • Our task was to identify the genetic defect in a mutant strain of C. elegans (SJ17) which apparently is unable to mount an unfolded protein response.

  4. The Unfolded Protein Response (UPR) XBP-1 IRE-1 • The UPR occurs when proteins are misfolded in the endoplasmic reticulum (ER). • Reducing agents, such as DTT, interfere with disulfide bond formation while tunicamysin interferes with glycosylation; both agents cause proteins to misfold in the ER thus triggering the UPR. • The product of the ire-1 gene is the sensor of misfolded proteins and when activated removes an intron from the pre mRNA from the xbp-1 gene. • Active xbp-1 protein (from spliced mRNA) activates the genes that code for ER chaperones, such as hsp-4.

  5. Experimental Design • Can we detect the UPR in the C. elegans? • Can the UPR be induced by Tunicamycin which causes ER stress? • Can the UPR be induced in the SJ17 mutants • Is there a survival phenotype for the SJ17 mutation? • Are SJ17 worms hypersensitive to tunicamysin? • Is the mutation located in ire-1 or xbp-1? • Does electrophoresis of the RT-PCR product show that it is xbp-1 mRNA is spliced or unspliced in mutants exposed to ER stress • If it is not spliced then the mutation is located in the ire-1. • If it is spliced then the mutation may lie in the xbp-1 gene.

  6. Mutant C. elegans do not Induce GFP:Hsp-4 in Response to Tunicamycin Untreated Control WILD TYPE MUTANT TunicamysinTreated WILD TYPE MUTANT

  7. 150 150 130 130 110 110 Total Counted Total Counted 90 90 # Eggs # Eggs L1-L2 L1-L2 70 70 L3-L4 L3-L4 Dead Dead 50 50 30 30 10 10 Day 1 Day 2 Day 3 Day 4 Day 1 Day 2 Day 3 Day 4 -10 -10 Date Observed Date Observed Survivorship of Wild Type C. elegans Figure 1- untreated wild type Figure 2- treated wild type

  8. Survivorship for Mutant C.elegans 150 150 130 130 110 Total Counted 110 Total Counted 90 90 # Eggs L1-L2 # Eggs 70 L3-L4 L1-L2 70 Dead L3-L4 50 Dead 50 30 30 10 10 Day 1 Day 2 Day 3 Day 4 -10 Day 1 Day 2 Day 3 Day 4 Date Observed -10 Date Observed Figure 3. Untreated Mutant Figure 4. Treated Mutant

  9. RT-PCR Analysis of Xbp-1 messenger RNA splicing In order to determine whether the Xbp-1 mRNA has been spliced we must: 1. Extract total RNA from our worms. 2. Determine the quality and quantity of the RNA extracted. 3. Amplify the xbp-1 mRNA sequence present in the samples. 4. Examine the amplified RNA for spliced (small) and unspliced (large) xpb-1 message. Figure 2:Electrophoresis of the RT-PCR product from the C. elegans extracted RNA. Figure 1:Electrophoresis of the RNA that was extracted from the C. elegans. Table 1: Lane identification key 1 2 3 4 5 1 2 3 4 5 400 bp UnsplicedSpliced 200 bp Unspliced – 294 bpSpliced – 271 bp This Electrophoresis Result indicates that RNA has been successfully extracted from our C. elegans. The Xbp-1 mRNA is spliced in both mutant and control animals exposed to Tunicamycin.

  10. Summary • We have shown that the UPR can be detected by using a fluorescent reporter protein (hsp4). The ER stressor, Tunicamycin, induces the GFP fusion hsp-4 product in wild type C. elegans, while in the SJ17 mutant, induction does not occur. • We have also shown that the phenotype for the mutant C. elegans (SJ17) exposed to tunicamysin, is a severe delay in growth that can result in death. • After careful analysis we have determined the mutation in SJ17 to be located in the xbp-1 gene because our RT-PCR analysis showed that splicing of xbp-1 mRNA occurred and therefore the ire-1 gene product which is responsible for splicing is functional.

More Related