Mechanism of brush border contractility studied by the quick-freeze, deep-etch method

The contractile behavior of intestinal brush borders remains poorly understood. Prior research has shown that epithelial cells contain terminal webs composed of actin filaments and cross-linking proteins. However, the exact mechanism of brush border contraction has not been fully resolved. Earlier studies have demonstrated that microvilli can change shape in response to ATP and temperature. Yet, the specific role of actin and myosin in this process remains unclear. No prior work had resolved how the terminal web contracts at the level of the zonula adherens. This gap motivated the use of high-resolution imaging techniques to observe structural changes during contraction. The absence of detailed ultrastructural data on filament arrangement and sliding has limited progress in this field. Understanding these mechanisms is essential for clarifying how epithelial cells regulate surface area and transport functions. This study aimed to address these unresolved questions using advanced electron microscopy.

The goal of this work was to investigate the mechanism of brush border contraction in intestinal epithelial cells. Researchers focused on the terminal web region, particularly near the zonula adherens. They sought to determine how ATP and temperature affect structural changes in this region. The study aimed to clarify whether actin and myosin filaments slide during contraction. They also wanted to identify the role of cross-linkers between microvillus rootlets. The use of glycerinated sheets and isolated brush borders allowed for controlled observations. The researchers hypothesized that filament sliding would be a key component of contraction. This work aimed to provide a detailed ultrastructural analysis of these processes.

The study used glycerinated chicken small intestine epithelium and isolated brush borders. Quick-freeze, deep-etch, and rotary shadow replication techniques were employed to capture ultrastructural details. These methods allowed for high-resolution imaging of filament arrangements. Mg-ATP was introduced at 37 degrees Celsius to induce contraction. The terminal web region was examined for structural changes near the zonula adherens. Myosin S1 decoration was used to identify actin filaments in circumferential bundles. Immunofluorescent labeling of myosin helped distinguish filament types. The researchers observed changes in filament diameter and cross-linker density during contraction.

The terminal web region constricted at the zonula adherens when exposed to Mg-ATP. This led to rounding of brush borders and splaying of microvilli into fanlike patterns. The most notable change was a decrease in the diameter of the circumferential ring of filaments. Cross-linkers between microvillus rootlets also decreased in number. Microvilli were not retracted into the terminal web during contraction. Myosin S1 decoration showed that most filaments in the bundle were actin. Some filaments lacked decoration and had projections attached to actin filaments. These undecorated filaments were proposed to be myosin based on immunofluorescent localization.

The study concludes that brush border contraction is primarily driven by sliding of actin and myosin filaments. This sliding occurs within the circumferential bundle near the zonula adherens. The decrease in filament diameter and cross-linker density supports this mechanism. The presence of undecorated filaments with myosin-like projections was noted. These findings align with the authors' hypothesis about filament interaction during contraction. The results suggest that filament polarity and arrangement are crucial for contraction. The absence of microvillus retraction indicates a different mechanism of structural change. These conclusions are directly based on the observed ultrastructural changes.