Abstract

By utilizing a combination of several ultrastructural techniques, we have been able to demonstrate differences in filament organization on the adherent plasma membranes of spreading and mobile PMN as well as within the extending lamellipodia. To follow the subplasmalemmal filaments of this small amoeboid cell during these kinetic events, we sheared off the upper portions of cells onto glass and carbon surfaces for 30 s--5 min. The exposed adherent membranes were immediately fixed and processed for high-resolution SEM or TEM. Whole cells were also examined by phase contrast microscopy, SEM, and oriented thin sections. Observed by SEM, the inner surface of nonadherent PMN membranes is free of filaments, but within 30 s of attachment to the substrate a three-dimensional, interlocking network of globular projections and radiating microfilaments--i.e., a subplasmalemmal filament complex--is consistently demonstrable (with or without postfixation in OsO4). Seen by TEM, extending lamellipodia contain a felt of filamentous and finely granular material, distinct from the golbule/filament complex of the adjacent adherent membrane. In the spread cell, this golbule-filament complex covers the entire lower membrane and increases in filament-density over the next 2--3 min. By 3--5 min after plating, as the PMN rounds up before the initiation of amoeboid movements, another pattern emerges--circumferential bands of anastomosing filament bundles in which thick, short filaments resembling myosin are found. This work provides structural evidence on the organization of polymerized contractile elements associated with the plasma membrane during cellular adherence.

Highlights

  • By utilizing a combination of several ultrastructural techniques, we have been able to demonstrate differences in filament organization on the adherent plasma membranes of spreading and mobile polymorphonuclear leukocytes (PMN) as well as within the extending lamellipodia

  • Circulating heterophilic polymorphonuclear leukocytes (PMN) must adhere to vessel walls before they can emigrate into tissues where they function as phagocytes

  • Parallel samples of adhering PMN were scrutinized by phase-contrast microscopy and fixed for conventional thin-sectional transmission electron microscopy (TEM) and whole-cell scanning electron microscopy (SEM), so that the structures observed on the exposed plasma membranes could be correlated with the more common aspects of cellular ultrastructure and specific cellular activities

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Summary

Introduction

By utilizing a combination of several ultrastructural techniques, we have been able to demonstrate differences in filament organization on the adherent plasma membranes of spreading and mobile PMN as well as within the extending lamellipodia. This work provides structural evidence on the organization of polymerized contractile elements associated with the plasma membrane during cellular adherence. After rupturing the amoebae with a shearing jet of buffered salt solution, these investigators were able to demonstrate actin-containing filaments on the exposed cytoplasmic surfaces of adherent membranes. Parallel samples of adhering PMN were scrutinized by phase-contrast microscopy and fixed for conventional thin-sectional TEM and whole-cell SEM, so that the structures observed on the exposed plasma membranes could be correlated with the more common aspects of cellular ultrastructure and specific cellular activities

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