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Diaphanous-1 affects the nanoscale clustering and lateral diffusion of receptor for advanced glycation endproducts
1Department of Chemistry, Iowa State University, Ames, IA 50011, United States.
Abstract:
The interactions between the cytoplasmic protein diaphanous-1 (Diaph1) and the receptor for advanced glycation endproducts (RAGE) drive the negative consequences of RAGE signaling in several disease processes. Reported in this work is how Diaph1 affects the nanoscale clustering and diffusion of RAGE measured using super-resolution stochastic optical reconstruction microscopy (STORM) and single particle tracking (SPT). Altering the Diaph1 binding site has a different impact on RAGE diffusion compared to when Diaph1 expression is reduced in HEK293 cells. In cells with reduced Diaph1 expression (RAGE-Diaph1-/-), the average RAGE diffusion coefficient is increased by 35%. RAGE diffusion is known to be influenced by the dynamics of the actin cytoskeleton. Actin labeling shows that a reduced Diaph1 expression leads to cells with reduced filopodia density and length. In contrast, when two RAGE amino acids that interact with Diaph1 are mutated (RAGERQ/AA), the average RAGE diffusion coefficient is decreased by 16%. Since RAGE diffusion is slowed when the interaction between Diaph1 and RAGE is disrupted, the interaction of the two proteins results in faster RAGE diffusion. In both RAGERQ/AA and RAGE-Diaph1-/- cells the number and size of RAGE clusters are decreased compared to cells expressing RAGE and native concentrations of Diaph1. This work shows that Diaph1 has a role in affecting RAGE clusters and diffusion.
Insights
Diaphanous-1 (Diaph1) protein influences the movement and clustering of the receptor for advanced glycation endproducts (RAGE). Disrupting Diaph1 binding or reducing its expression alters RAGE diffusion and reduces RAGE cluster size.
Area of Science:
- Cell biology
- Molecular interactions
- Biophysics
Background:
- The receptor for advanced glycation endproducts (RAGE) is implicated in various disease processes.
- RAGE signaling is influenced by interactions with cytoplasmic proteins.
- Diaphanous-1 (Diaph1) is a key protein interacting with RAGE.
Purpose of the Study:
- To investigate how Diaph1 affects the nanoscale clustering and diffusion of RAGE.
- To elucidate the role of the Diaph1-RAGE interaction in RAGE dynamics.
Main Methods:
- Super-resolution stochastic optical reconstruction microscopy (STORM).
- Single particle tracking (SPT) to measure RAGE diffusion.
- Actin cytoskeleton labeling.
- HEK293 cells with altered Diaph1 expression or RAGE-Diaph1 binding sites.
Main Results:
- Reduced Diaph1 expression increased RAGE diffusion by 35% and decreased filopodia.
- Mutating Diaph1 binding sites on RAGE decreased RAGE diffusion by 16%.
- Both conditions reduced the number and size of RAGE clusters.
Conclusions:
- Diaph1 binding promotes faster RAGE diffusion.
- Diaph1 influences RAGE dynamics and nanoscale organization.
- The Diaph1-RAGE interaction plays a significant role in RAGE clustering and movement.
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