The intramembrane structure of septate junctions based on direct freezing

Area of Science:

• Cell Biology
• Biophysics
• Zoology

Background:

• Septate junctions are crucial for maintaining paracellular barrier function in invertebrates.
• Previous studies on septate junction structure have relied on fixation methods that may introduce artifacts.

Purpose of the Study:

• To investigate the ultrastructure of smooth and Hydra-type septate junctions using direct freeze-fracture.
• To compare the observed structures with those obtained from conventional fixation techniques.

Main Methods:

• Direct freeze-fracture using liquid helium-cooled copper block/slam freezing.
• Analysis of exoplasmic and protoplasmic fracture faces.
• Comparison with freeze-fracture replicas of fixed tissues and lanthanum-infiltrated sections.

Main Results:

• Both smooth (Acheta, Limulus) and Hydra-type septate junctions exhibit rows of irregular intramembrane particles on the exoplasmic face.
• Protoplasmic faces show complementary grooves with periodic variations, more pronounced in Hydra.
• Observed structures differ significantly from those in fixed and cryoprotected tissues, highlighting potential artifacts.

Conclusions:

• Direct freeze-fracture reveals native septate junctional structures, likely representing deformed transmembrane proteins.
• The study identifies artifacts associated with glutaraldehyde fixation and cryoprotection in freeze-fracture electron microscopy.
• Provides a clearer understanding of intramembrane components and their relation to intercellular septal elements.