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Intracellular pH modulation of ADF/cofilin proteins
B W Bernstein1, W B Painter, H Chen
1Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA. bwb@lamar.colostate.edu
This study explores how intracellular pH (pH(i)) affects ADF/cofilin (AC) proteins in Swiss 3T3 cells. AC proteins help regulate actin filament turnover, a process crucial for cell movement and shape changes. The researchers found that pH(i) influences where AC proteins are located in the cell and how they interact with actin. At higher pH(i), ADF moves more to monomeric actin, while cofilin distribution remains stable. Only a specific mutant of AC (A3) shows unique cellular effects when pH(i) changes. The study also shows that phospho-AC levels increase with cell density and motility, suggesting a regulatory mechanism. These findings suggest that pH(i) plays a key role in modulating AC activity in living cells.
Area of Science:
- Cellular signaling pathways in biochemistry
- Cytoskeletal regulation in cell biology
- Actin dynamics in molecular physiology
Background:
Cells regulate actin filament turnover to adapt to stimuli. ADF/cofilin (AC) proteins are key players in this process. Phosphorylation and pH <7.1 inhibit AC activity in vitro. However, how intracellular pH (pH(i)) affects AC in living cells remains unclear. Prior research has shown pH influences actin dynamics, but the specific role of pH(i) in AC behavior is not fully understood. This gap motivated the study of pH(i) effects on AC localization and activity in Swiss 3T3 cells. No prior work had resolved how pH(i) alters AC partitioning with actin. The study aimed to clarify pH(i)-dependent AC behavior in a physiological context. Understanding this could reveal how cells modulate actin turnover in response to environmental changes. The findings may help explain pH-regulated cytoskeletal remodeling in motile cells.
Purpose Of The Study:
This study aimed to determine how intracellular pH (pH(i)) influences ADF/cofilin (AC) protein behavior in vivo. The researchers focused on pH(i)-dependent AC translocation and activity in Swiss 3T3 cells. They sought to clarify whether pH(i) affects AC partitioning with actin monomers or polymers. The study also explored how pH(i) influences the effects of phosphorylation on AC function. The researchers hypothesized that pH(i) modulates AC activity in a manner similar to in vitro observations. They wanted to test if pH(i) could alter AC localization to membranes during cell stimulation. The study aimed to identify if pH(i) changes could lead to distinct cellular phenotypes when AC is mutated. The findings could help explain how pH(i) regulates cytoskeletal dynamics in living cells.
Main Methods:
The study used Swiss 3T3 cells to investigate pH(i)-dependent AC behavior. Intracellular pH was manipulated using pH buffers to alter pH(i) between 6.6 and 7.4. Researchers monitored AC translocation to ruffling membranes after wounding cells. Immunostaining was used to analyze AC partitioning with monomeric and polymeric actin. Covariance and colocalization analyses were performed to assess AC distribution. The team overexpressed wild-type and unphosphorylatable AC mutants fused with GFP. Cellular phenotypes were observed under pH(i) shifts from 7.1 to 6.6 or 7.4. The effects of pH(i) on phospho-AC levels were measured in relation to cell density and motility. The study combined biochemical assays with live-cell imaging to track AC behavior.
Main Results:
AC translocated to ruffling membranes in wounded cells only at alkaline pH(i). Soluble ADF decreased from 42±4% to 23±4% when pH(i) dropped from 7.4 to 6.6. ADF partitioned more with monomeric actin and less with polymeric actin as pH(i) increased. Cofilin distribution remained unchanged with pH(i) variations. Only the unphosphorylatable AC mutant (A3) produced aberrant phenotypes when pH(i) shifted. AC(A3)-GFP and AC(wt)-GFP showed distinct behaviors under pH(i) changes. Phospho-AC levels increased with cell density and motility. Alkalization of pH(i) in motile cells also increased phospho-AC levels. These findings suggest pH(i) modulates AC activity in a homeostatic manner.
Conclusions:
The study shows that intracellular pH (pH(i)) affects ADF/cofilin (AC) behavior in vivo. AC translocation to membranes and partitioning with actin depend on pH(i). Phosphorylation and pH(i) together regulate AC activity in living cells. The unphosphorylatable AC mutant (A3) produced unique phenotypes under pH(i) shifts. Phospho-AC levels increased with cell density and motility, suggesting a homeostatic mechanism. Alkalization of pH(i) in motile cells increased phospho-AC levels. These findings support the idea that pH(i) modulates AC activity in a regulated manner. The results align with in vitro observations of pH(i)-dependent AC inhibition.
Frequently Asked Questions
AC proteins translocate to membranes and partition with actin differently depending on pH(i). At pH 7.4, ADF partitions more with monomeric actin.
Phosphorylation inhibits AC activity in vitro. In vivo, phospho-AC levels increase with pH(i) and cell motility, suggesting a regulatory role.
The A3 mutant (unphosphorylatable) shows aberrant behavior only when pH(i) shifts from 7.1 to 6.6 or 7.4.
ADF partitions more with monomeric actin at higher pH(i), while cofilin distribution remains unchanged.
Phospho-AC levels rise with cell density and motility, suggesting a homeostatic mechanism to regulate AC activity.
The study proposes that pH(i) modulates AC activity in a homeostatic manner to balance actin turnover in cells.