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Indirect immunofluorescence localization of ponticulin in motile cells
L J Wuestehube1, C P Chia, E J Luna
1Department of Biology, Princeton University, New Jersey.
Abstract:
Ponticulin is the major actin-binding integral glycoprotein in plasma membranes isolated from log-phase Dictyostelium discoideum amebae. As such, this protein appears to be an important link between the plasma membrane and actin filaments (Wuestehube and Luna: Journal of Cell Biology 105:1741-1751, 1987). In this study, indirect immunofluorescence microscopy was used to examine the distribution of ponticulin in randomly moving D. discoideum amebae and in amebae engaged in cell migration and phagocytosis. Ponticulin is distributed throughout the plasma membrane and also is present in intracellular vesicles associated with the microtubule-organizing center-Golgi complex adjacent to the nucleus. In aggregating amebae, ponticulin is concentrated in regions of lateral cell-cell contact and in arched regions of the plasma membrane. Ponticulin also is present, but not obviously enriched, in filopodia, in the actin-rich anterior end of polarized cells, and in detergent-insoluble cytoskeletons. In amebae engaged in phagocytosis of yeast, ponticulin is present but not enriched in phagocytic cups and is associated with intracellular vesicles around engulfed yeast. These results suggest that ponticulin is stably associated with actin filaments in certain regions of the plasma membrane and that the actin-binding activity of ponticulin may be tightly controlled. Indirect immunofluorescence microscopy and immunoblot analysis demonstrate that human polymorphonuclear leukocytes also contain a 17 kD protein that specifically cross-reacts with antibodies affinity-purified against D. discoideum ponticulin. As in D. discoideum, the mammalian 17 kD ponticulin-analog appears to be localized in plasma membrane and is evident in actin-rich cell extensions. These results indicate that ponticulin-mediated linkages between the plasma membrane and actin may be present in higher eukaryotic cells.
Insights
Ponticulin, a major actin-binding protein in Dictyostelium discoideum, links the plasma membrane to actin filaments. A similar protein exists in human leukocytes, suggesting conserved roles in cell structure and migration.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Ponticulin is a key actin-binding glycoprotein in Dictyostelium discoideum plasma membranes.
- It acts as a crucial link between the plasma membrane and the actin cytoskeleton.
- Its precise distribution and regulation in dynamic cellular processes are not fully understood.
Purpose of the Study:
- To investigate the subcellular distribution of ponticulin in Dictyostelium discoideum during various cellular activities.
- To explore the presence and localization of ponticulin homologs in mammalian cells.
- To understand the functional implications of ponticulin-actin interactions.
Main Methods:
- Indirect immunofluorescence microscopy was employed to visualize ponticulin distribution.
- Immunoblot analysis was used to detect cross-reacting proteins in human leukocytes.
- Detergent-insolubility assays were performed on cytoskeletons.
Main Results:
- Ponticulin is found throughout the plasma membrane and in intracellular vesicles in Dictyostelium.
- It concentrates at cell-cell contacts and in arched membrane regions during aggregation.
- A 17 kD ponticulin-analog was identified in human polymorphonuclear leukocytes, localized to the plasma membrane and cell extensions.
Conclusions:
- Ponticulin exhibits dynamic localization patterns, suggesting regulated association with actin filaments.
- The presence of a ponticulin-analog in human leukocytes indicates conserved ponticulin-mediated plasma membrane-actin linkages in higher eukaryotes.
- These findings highlight the potential importance of ponticulin in fundamental cellular processes across different organisms.