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Published on: December 3, 2019
Permeability changes of Manduca sexta midgut brush border membranes induced by oligomeric structures of different cry
C Muñoz-Garay1, J Sánchez, A Darszon
1Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México.
Abstract:
The pore-formation activity of monomeric and oligomeric forms of different Cry1 toxins (from Cry1A to Cry1G) was analyzed by monitoring ionic permeability across Manduca sexta brush border membrane vesicles. The membrane vesicles were isolated from microvilli structures, showing a high enrichment of apical membrane markers and low intrinsic K(+) permeability. A fluorometric assay performed with 3,3'-dipropylthiodicarbocyanine fluorescent probe, sensitive to changes in membrane potential, was used. Previously, it was suggested that fluorescence determinations with this dye could be strongly influenced by the pH, osmolarity and ionic strength of the medium. Therefore, we evaluated these parameters in control experiments using the K(+)-selective ionophore valinomycin. We show here that under specific ionic conditions changes in fluorescence can be correlated with ionic permeability without effects on osmolarity or ionic strength of the medium. It is extremely important to attenuate the background response due to surface membrane potential and the participation of the endogenous permeability of the membrane vesicles. Under these conditions, we analyzed the pore-formation activity induced by monomeric and oligomeric structures of different Cry1 toxins. The Cry1 toxin samples containing oligomeric structures correlated with high pore activity, in contrast to monomeric samples that showed marginal pore-formation activity, supporting the hypothesis that oligomer formation is a necessary step in the mechanism of action of Cry toxins.
Insights
Cry toxin pore formation requires oligomerization. Studies show oligomeric Cry1 toxins exhibit significant pore activity, unlike monomeric forms, supporting their essential role in the Cry toxin mechanism of action.
Area of Science:
- Biochemistry
- Molecular Biology
- Insect Toxicology
Background:
- Cry toxins are pore-forming proteins crucial for insecticidal activity.
- Understanding the mechanism of Cry toxin pore formation is vital for developing new pest control strategies.
- Previous studies suggested oligomerization as a key step, but direct evidence was limited.
Purpose of the Study:
- To investigate the pore-formation activity of monomeric and oligomeric Cry1 toxins.
- To validate a fluorometric assay for measuring ionic permeability across insect membrane vesicles.
- To confirm the necessity of oligomer formation for Cry toxin pore activity.
Main Methods:
- Isolation of Manduca sexta brush border membrane vesicles enriched in apical markers.
- Utilizing a 3,3'-dipropylthiodicarbocyanine fluorescent probe assay to monitor membrane potential changes.
- Performing control experiments to account for pH, osmolarity, and ionic strength influences on fluorescence.
- Analyzing pore formation by comparing ionic permeability induced by monomeric and oligomeric Cry1 toxin structures.
Main Results:
- Established specific ionic conditions for reliable fluorescence-based ionic permeability measurements.
- Demonstrated that oligomeric Cry1 toxins exhibit significantly higher pore-formation activity than monomeric forms.
- Showed that monomeric Cry1 toxins possess only marginal pore-formation activity.
Conclusions:
- Cry toxin oligomerization is a necessary prerequisite for pore formation.
- The findings support the hypothesis that oligomer formation is a critical step in the Cry toxin mechanism of action.
- This study provides robust evidence for the role of oligomeric structures in Cry toxin-mediated membrane permeabilization.
