The structure of the contractile apparatus in ultrarapidly frozen smooth muscle: freeze-fracture, deep-etch, and freeze-substitution studies

Abstract

The structure of the smooth muscle contractile apparatus was studied using ultrarapid freezing followed by freeze substitution or by longitudinal freeze-fracture, deep-etch, and platinum-carbon replication. Freeze substitution minimises the detrimental effects of chemical fixation and freeze fracture eliminates them entirely whilst revealing the ultrastructure in three dimensions. Unidirectionally shadowed freeze-fracture replicas of ultrarapidly frozen, relaxed, intact smooth muscle showed a well-preserved actin filament structure the 5.5-nm repeat of the actin subunits was clearly observed. In transversely fractured tissue the thick filaments were revealed, with a distribution comparable to that seen in transverse sections of freeze-substituted muscle. Relaxed muscle permeabilised using Triton X-100 showed a similar structure to that of intact tissue after ultrarapid freezing and examination both by freeze fracture and by freeze examination both by freeze fracture and by freeze substitution; the ratios of actin to myosin were also comparable. In permeabilised, rigorised tissue the structure of the actomyosin complex was revealed in detail; this was especially clear in freeze-substituted muscle. A cross-bridge spacing of 38 nm was measured in freeze-fractured, deep-etched tissue. The structural detail revealed is compatible with a side polar model of the actomyosin interaction and with the sliding filament mechanism of muscle contraction.