Detail of 100ms_ThT_1.5x_40x_ND8_1_2_Pos7
Project
Title
Fluorescence images of thioflavin-T with mCherry-Nter (N-terminal region of Argonaute2) in the absence of seeds of Nter aggregates.
Description
Fluorescence images of thioflavin-T, an amyloid detection reagent, with mCherry-Nter (N-terminal region of Argonaute2) in the absence of seeds of Nter aggregates. In the absence of the seeds, the ThT fluorescence intensity showed an initial lag phase of 300 min and then reached a plateau at 800 min after mixing monomeric Nter with ThT in aggregation buffer.
Release, Updated
2024-12-23
License
CC-BY
Kind
Image data
File Formats
.tif
Data size
722.7 MB
Datatype
-
Molecular Function (MF)
Biological Process (BP)
protein-containing complex assembly
(
GO:0065003
)
Cellular Component (CC)
Biological Imaging Method
X scale
0.108 micrometer/pixel
Y scale
0.108 micrometer/pixel
Z scale
-
T scale
10 minutes per time interval
Image Acquisition
Experiment type
-
Microscope type
-
Acquisition mode
-
Contrast method
-
Microscope model
-
Detector model
-
Objective model
-
Filter set
-
Summary of Methods
See details in Narita H, et. al. BMC Biol. 2023 Apr 19;21(1):78.
Related paper(s)
Haruka Narita, Tomohiro Shima, Ryo Iizuka, Sotaro Uemura (2023) N-terminal region of Drosophila melanogaster Argonaute2 forms amyloid-like aggregates., BMC biology, Volume 21, Number 1, pp. 78
Published in 2023 Apr 19 (Electronic publication in April 19, 2023, midnight )
- doi: 10.1186/s12915-023-01569-3
-
pmid: 37072852
(Abstract) BACKGROUND: Argonaute proteins play a central role in RNA silencing by forming protein-small RNA complexes responsible for the silencing process. While most Argonaute proteins have a short N-terminal region, Argonaute2 in Drosophila melanogaster (DmAgo2) harbors a long and unique N-terminal region. Previous in vitro biochemical studies have shown that the loss of this region does not impair the RNA silencing activity of the complex. However, an N-terminal mutant of Drosophila melanogaster has demonstrated abnormal RNA silencing activity. To explore the causes of this discrepancy between in vitro and in vivo studies, we investigated the biophysical properties of the region. The N-terminal region is highly rich in glutamine and glycine residues, which is a well-known property for prion-like domains, a subclass of amyloid-forming peptides. Therefore, the possibility of the N-terminal region functioning as an amyloid was tested. RESULTS: Our in silico and biochemical assays demonstrated that the N-terminal region exhibits amyloid-specific properties. The region indeed formed aggregates that were not dissociated even in the presence of sodium dodecyl sulfate. Also, the aggregates enhanced the fluorescence intensity of thioflavin-T, an amyloid detection reagent. The kinetics of the aggregation followed that of typical amyloid formation exhibiting self-propagating activity. Furthermore, we directly visualized the aggregation process of the N-terminal region under fluorescence microscopy and found that the aggregations took fractal or fibril shapes. Together, the results indicate that the N-terminal region can form amyloid-like aggregates. CONCLUSIONS: Many other amyloid-forming peptides have been reported to modulate the function of proteins through their aggregation. Therefore, our findings raise the possibility that aggregation of the N-terminal region regulates the RNA silencing activity of DmAgo2.(MeSH Terms)
Contact
Tomohiro Shima, Sotaro Uemura
, University of Tokyo, University of Tokyo
, Department of Biological Sciences, Graduate School of Science , Department of Biological Sciences, Graduate School of Science
Contributors
Haruka Narita
OMERO Project
Source