Localization of putative membrane raft proteins involved in Caenorhabditis elegans’ immune response to Stenotrophomonas maltophilia.
Eric Nelson
Author
04/02/2021
Added
20
Plays
Description
This study uses CRISPR/Cas9 technology to create GFP translational fusion proteins to to determine the localization and expression patterns of the putative membrane raft proteins ZK6.11, DOD-19, and B0024.4 in C. elegans when exposed to pathogenic S. maltophilia.
Searchable Transcript
Toggle between list and paragraph view.
- [00:00:00.870]Hello everyone. My name is Eric Nelson.
- [00:00:03.270]The title of my project is the "Localization of putative membrane raft proteins
- [00:00:07.140]involved in C. elegans immune response to Stenotrophomonas maltophilia."
- [00:00:11.970]I've been running my research under the guidance of Dr. Michael Herman,
- [00:00:14.970]and Leah Radeke in the school of biological sciences here at UNL.
- [00:00:20.310]To start, I would like to give you some background information on my study.
- [00:00:23.940]Stenotrophomonas maltophilia is an emerging gram-negative, nosocomial
- [00:00:27.780]pathogen that can cause respiratory infections in immunocompromised patients.
- [00:00:33.330]Stenotrophomonas is part of the microbiome and the natural environment of
- [00:00:37.050]C. elegans, making it an excellent model organism to study pathogen-host
- [00:00:41.490]interactions and the innate immune system.
- [00:00:44.640]The Herman lab has previously previously identified genes in C. elegans
- [00:00:48.710]involved in response to pathogenic S. maltophilia. Of these genes
- [00:00:53.460]identified, ZK6.11, dod-19, and B0024.4
- [00:00:58.410]are all glycoproteins chosen for further analysis due to their
- [00:01:02.820]involvement in the immune response to pathogenic S. maltophilia and their
- [00:01:07.350]association with the membrane raft. Further,
- [00:01:10.350]mutants of these glycoproteins showed increased susceptibility to
- [00:01:15.030]pathogenic S. maltophila. As you can see here,
- [00:01:18.810]this figure demonstrates the pathogenicity of E. coli OP50
- [00:01:22.140]and S. maltophilia strains on C. elegans worms. When exposed
- [00:01:27.220]to each strain, worms on pathogenic strains
- [00:01:30.180]JV3 and JCMS lived about half as long as the less pathogenic
- [00:01:35.340]K279a and OP50.
- [00:01:40.290]Here we have a heat map showing the relative survivals of
- [00:01:44.100]B0024.4,
- [00:01:46.260]ZK6.11, and dod-19 mutants on OP50,
- [00:01:51.270]K279a, JCMS, and JV3,
- [00:01:54.660]all compared to the wild type. Blue squares demonstrate that mutants for
- [00:01:59.430]those genes survive longer than normal,
- [00:02:02.070]whereas red squares demonstrate shorter lifespans. Here,
- [00:02:05.580]You can see that lifespan of
- [00:02:07.710]ZK6.11 and dod-19 were significantly reduced on JCMS,
- [00:02:12.390]Whereas B0024.4 had a significantly shorter life span on JV3.
- [00:02:17.100]We know these genes are interesting because of these reasons,
- [00:02:21.240]And so now we want to study them further and see their expression patterns.
- [00:02:25.620]This study aims to determine the localization and expression patterns of the
- [00:02:29.220]putative membrane raft proteins ZK6.11, DOD-19,
- [00:02:33.030]and B0024.4 in C. elegans
- [00:02:37.020]when exposed to pathogenic S. maltophilia. To do this,
- [00:02:40.950]this study uses CRISPR/Cas9 to incorporate the green fluorescent protein at
- [00:02:45.480]the beginning or end of the three genes.
- [00:02:48.510]This figure shows a gRNA guiding Cas9 protein to cut double
- [00:02:53.250]stranded organismal DNA at a defined locus.
- [00:02:57.000]The injected organism then uses homology directed repair using
- [00:03:02.170]the GFP destination vector,
- [00:03:04.330]which incorporates the GFP tag, modifying the translated protein to now
- [00:03:09.100]carry a GFP tag as well.
- [00:03:13.450]This study uses CRISPR/Cas9 to create translational fusions containing the
- [00:03:17.500]protein of interest fused to green fluorescent protein.
- [00:03:20.890]This process involves several steps:
- [00:03:23.890]We used the SapTrap method to design and create a destination vector with green
- [00:03:28.750]fluorescent protein and a gRNA to tag each gene.
- [00:03:33.580]Next, we injected the destination vector,
- [00:03:36.010]a Cas9 expressing vector, and two negative selection markers
- [00:03:40.870]into young adult C. elegans gonads. Next,
- [00:03:45.550]the offspring of the injected worms are then screened for GFP expression and
- [00:03:49.750]expression of other positive and negative selection markers.
- [00:03:53.560]The negative selection markers help to differentiate whether the GFP
- [00:03:57.280]marker was properly integrated into the genome,
- [00:04:00.280]or is in worms in the form of an extrachromosomal array.
- [00:04:04.540]If the DNA is integrated,
- [00:04:06.010]then the negative selection markers would not be visible in the worms.
- [00:04:10.000]If the DNA is not integrated,
- [00:04:11.560]then the negative selection markers will be visible. Finally,
- [00:04:16.270]the GFP tagged worms will be exposed to OP50, K279a,
- [00:04:20.800]JV3, or JCMS to look for their expression patterns and cellular
- [00:04:25.690]and subcellular localizations.
- [00:04:29.680]This study is not completed yet, but has a lot of progress already made.
- [00:04:34.550]For ZK6.11 and B0024.4,
- [00:04:37.630]I'm currently in the process of constructing the destination vectors in vitro.
- [00:04:43.000]For DOD-19,
- [00:04:43.930]I'm currently in the process of injecting the constructed destination vector into
- [00:04:48.310]C. elegans and screening for the selection markers.
- [00:04:53.200]Because we know that these genes localize to the membrane raft,
- [00:04:56.620]these analyses will help to elucidate the role of membrane rafts in the immune
- [00:05:00.670]response to pathogens such as S. maltophilia and may play a role in
- [00:05:05.200]common innate immune pathways
- [00:05:08.650]such as the DAF-2/16 pathway or others.
- [00:05:12.760]These analyses can also identify novel gene functions in innate immune
- [00:05:17.650]signaling in C. elegans. That could be conserved across organisms.
- [00:05:23.260]I would like to thank the following groups for their assistance into this
- [00:05:26.350]project: the Herman lab, especially
- [00:05:29.170]Dr. Michael Herman and Leah Radeke for their assistance and intellectual
- [00:05:32.650]contributions.
- [00:05:34.240]The University of Nebraska-Lincoln, School of Biological Sciences for supporting
- [00:05:38.530]my research and for helping to put on this research fair and UNL UCARE
- [00:05:43.270]grant program for its financial support in my research and development.
- [00:05:47.980]Thank you.
The screen size you are trying to search captions on is too small!
You can always jump over to MediaHub and check it out there.
Log in to post comments
Embed
Copy the following code into your page
HTML
<div style="padding-top: 56.25%; overflow: hidden; position:relative; -webkit-box-flex: 1; flex-grow: 1;">
<iframe
style="bottom: 0; left: 0; position: absolute; right: 0; top: 0; border: 0; height: 100%; width: 100%;"
src="https://mediahub.unl.edu/media/16224?format=iframe&autoplay=0"
title="Video Player: Localization of putative membrane raft proteins involved in Caenorhabditis elegans’ immune response to Stenotrophomonas maltophilia."
allowfullscreen
></iframe>
</div>
Comments
0 Comments