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Changing patterns of plasma membrane-associated filaments during the initial phases of polymorphonuclear leukocyte
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.
Insights
Neutrophil (PMN) plasma membranes exhibit distinct filament organization during cell spreading and movement. Ultrastructural analysis reveals a dynamic subplasmalemmal filament complex crucial for cellular adherence and motility.
Area of Science:
- Cell Biology
- Biophysics
- Microscopy
Background:
- Understanding the dynamic structural changes in neutrophil (PMN) plasma membranes is essential for elucidating cellular adherence and motility.
- The subplasmalemmal cytoskeleton plays a critical role in cell shape regulation and movement.
Purpose of the Study:
- To investigate and demonstrate differences in filament organization on the adherent plasma membranes of spreading and mobile neutrophils (PMN).
- To characterize the subplasmalemmal filament complex and its structural changes during neutrophil adherence and lamellipodia extension.
Main Methods:
- Utilized a combination of ultrastructural techniques, including high-resolution Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM).
- Employed a cell shearing technique to expose adherent plasma membranes for immediate fixation and analysis.
- Examined whole cells using phase contrast microscopy and oriented thin sections.
Main Results:
- Nonadherent PMN membranes lack filaments; however, within 30 seconds of attachment, a three-dimensional network of globular projections and microfilaments (subplasmalemmal filament complex) forms.
- Extending lamellipodia display a distinct filamentous and granular material, differing from the adjacent adherent membrane complex.
- In spread cells, the filament complex covers the entire membrane, increasing in density over 2-3 minutes, with circumferential bands appearing before movement initiation.
Conclusions:
- Demonstrated distinct structural organization of subplasmalemmal filaments during neutrophil adherence, spreading, and preparation for movement.
- Provided structural evidence for the organization of polymerized contractile elements associated with the plasma membrane during cellular adherence.