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Localization and mobility of gelsolin in cells
J A Cooper1, D J Loftus, C Frieden
1Department of Biological Chemistry, Washington University School of Medicine, St. Louis Missouri 63110.
The Journal of Cell Biology
|April 1, 1988
Summary
Gelsolin is a protein found throughout the cytoplasm in living cells. In fixed cells, it appears associated with actin filaments, but it is not tightly bound to them.
Area of Science:
- Cell Biology
- Biochemistry
Background:
- Gelsolin is an actin-binding protein involved in regulating actin dynamics.
- Its precise localization and interaction with actin filaments in living cells remain incompletely understood.
Purpose of the Study:
- To investigate the physiologic role of gelsolin by studying its location and mobility in mouse fibroblast cells.
- To determine if gelsolin is tightly bound to actin filaments in living cells.
Main Methods:
- Immunofluorescence microscopy of fixed and permeabilized cells.
- Fluorescent analog cytochemistry of living cells.
- Fluorescence photobleaching recovery (FRAP) to measure gelsolin mobility.
- Immunoblotting to assess protein extraction.
Main Results:
- In living cells, gelsolin exhibits a diffuse cytoplasmic distribution.
- In fixed cells, a minor fraction of gelsolin associates with actin-rich regions, an effect more pronounced after permeabilization.
- Fluorescently labeled gelsolin microinjected into living cells is fully mobile, with a diffusion coefficient similar to control proteins.
- Fluorescent phalloidin, a known actin binder, is immobile, serving as a positive control.
- Endogenous gelsolin is rapidly extracted from detergent-permeabilized cells, consistent with diffusion.
Conclusions:
- Gelsolin is not tightly bound to actin filaments in living cells.
- The association observed in fixed cells likely represents transient interactions trapped during the fixation process.
- Gelsolin's mobility suggests a role in dynamic cellular processes rather than stable structural association with actin.