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Rho is a negative regulator of human monocyte spreading
M Aepfelbacher1, M Essler, E Huber
1Institute for Prevention of Cardiovascular Diseases, University of Munich, Germany.
Abstract:
Monocyte spreading is a sequential series of events including cell flattening, formation of new actin structures and focal adhesions, as well as development of cytoplasmic projections. To investigate the involvement of the GTP-binding protein Rho in spreading, we treated human blood monocytes or PMA-stimulated U937 or THP1 cells with Clostridium botulinum C3-transferase (C3), which ADP-ribosylates and inactivates Rho in intact cells. The C3 treatment caused 1) a four- to fivefold increase in the number of THP1 cells that spread on fibronectin within 24 h of PMA stimulation, 2) a greater area covered by the spread cells, and 3) accelerated and enhanced development of macrophage-like filopodial and pseudopodial projections. Similar results were obtained with PMA-stimulated U937 cells and human blood monocytes. Furthermore, cell staining revealed disorganization of subcortical actin in C3-treated THP1 cells, whereas circular actin formations at the substrate-attached part of the cells and vinculin-containing focal complexes/adhesions were unaffected. Finally, we found a decrease in membrane-associated RhoA in normal spreading THP1 cells, which suggests endogenous inactivation of Rho and might provide an explanation for the acceleration of spreading caused by the C3-transferase. In conclusion, these results indicate that active Rho is an important, negative regulator of human monocyte spreading by maintaining cell tension and cortical actin organization.
Insights
Inhibiting the Rho GTP-binding protein with C3-transferase significantly enhances monocyte spreading and projection formation. This suggests active Rho negatively regulates monocyte cell spreading.
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- Monocyte spreading is a complex process involving cell shape changes, actin remodeling, and focal adhesion formation.
- The GTP-binding protein Rho is known to play a role in regulating cell shape and cytoskeletal organization.
Purpose of the Study:
- To investigate the role of the Rho GTP-binding protein in the process of human monocyte spreading.
- To determine if inhibiting Rho activity affects monocyte morphology and cytoskeletal dynamics during spreading.
Main Methods:
- Treatment of human blood monocytes and PMA-stimulated U937/THP1 cells with Clostridium botulinum C3-transferase (C3) to inactivate Rho.
- Assessment of cell spreading, area coverage, and projection formation on fibronectin.
- Analysis of subcortical actin organization and focal adhesions using cell staining techniques.
Main Results:
- C3 treatment significantly increased the number and area of spread THP1 cells, U937 cells, and human monocytes.
- Macrophage-like filopodial and pseudopodial projections were accelerated and enhanced in C3-treated cells.
- Disorganization of subcortical actin was observed in C3-treated cells, while focal adhesions remained unaffected.
Conclusions:
- Active Rho GTP-binding protein acts as a negative regulator of human monocyte spreading.
- Rho likely maintains cell tension and cortical actin organization, thereby inhibiting excessive spreading.
- Endogenous Rho inactivation during normal spreading may explain the observed acceleration upon C3 treatment.