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Myosin light chain phosphorylation does not increase during yeast phagocytosis by macrophages
P de Lanerolle1, G Gorgas, X Li
1Department of Physiology and Biophysics, University of Illinois, Chicago 60612.
Abstract:
We have studied the role of myosin II light chain phosphorylation in yeast phagocytosis by J774 cells. J774 cells, which are mouse cells of monocyte/macrophage lineage, ingest opsonized yeast particles, and the rate of internalization is linear for 60 min at 37 degrees C. Immunoprecipitation of myosin II from cells labeled with 32P, using an affinity-purified antibody to myosin II purified from J774 cells, demonstrated phosphorylation of both the myosin heavy chain and the 20-kDa light chain (PMLC) prior to the addition of the opsonized yeast. However, the levels of heavy chain and PMLC phosphorylation did not change during the linear phase of yeast uptake by J774 cells. Other experiments demonstrated that the amount of myosin II associated with the cytoskeleton did not change during phagocytosis, further supporting the observation that PMLC phosphorylation does not increase during phagocytosis. In contrast, F-actin increased by 1.6-fold during the linear phase of phagocytosis. Two additional approaches were used to analyze in greater detail the role of myosin II phosphorylation in phagocytosis. First, antibodies to myosin light chain kinase (MLCK), the enzyme that phosphorylates PMLC, were electroinjected into J774 cells. These antibodies, which inhibit MLCK activity, inhibited chemotaxis as previously described but had no effect on phagocytosis. Second, quantitation of phagocytosis and chemotaxis following treatment with the phosphoprotein phosphatase inhibitor okadaic acid demonstrated that chemotaxis was much more sensitive than phagocytosis to okadaic acid treatment; at 0.3 microM okadaic acid, there is a substantial increase in myosin phosphorylation and chemotaxis is inhibited by 60%, whereas phagocytosis is unaffected. These data indicate that PMLC phosphorylation and, by implication, myosin II are not involved in yeast phagocytosis. They also suggest that PMLC phosphorylation displays a high degree of specificity with respect to mediating energy-dependent cellular processes in macrophages.
Insights
Myosin II light chain phosphorylation does not play a role in yeast phagocytosis by J774 macrophage cells. Studies show no change in phosphorylation during yeast uptake, indicating myosin II is not involved in this cellular process.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Phagocytosis is a critical cellular process for immune cells like macrophages.
- Myosin II, a motor protein, is involved in various cellular functions, including cell motility and cytokinesis.
- Phosphorylation of myosin II light chain (PMLC) is a known regulatory mechanism.
Purpose of the Study:
- To investigate the role of myosin II light chain phosphorylation in yeast phagocytosis by J774 macrophage cells.
- To determine if changes in PMLC phosphorylation correlate with the rate of phagocytosis.
- To assess the impact of inhibiting myosin light chain kinase (MLCK) on phagocytosis.
Main Methods:
- J774 cells were used to study yeast phagocytosis.
- Immunoprecipitation and 32P labeling were employed to measure PMLC phosphorylation.
- Antibodies against MLCK were introduced into cells to inhibit its activity.
- Okadaic acid, a phosphatase inhibitor, was used to modulate phosphorylation levels.
Main Results:
- PMLC phosphorylation levels did not change during the linear phase of yeast phagocytosis.
- Myosin II association with the cytoskeleton remained constant during phagocytosis.
- Inhibition of MLCK or treatment with okadaic acid did not affect phagocytosis, although chemotaxis was impacted.
- F-actin levels increased during phagocytosis, suggesting its involvement.
Conclusions:
- PMLC phosphorylation and myosin II are not involved in yeast phagocytosis by J774 cells.
- PMLC phosphorylation appears to be specific in mediating energy-dependent cellular processes in macrophages.
- These findings highlight the complexity of cellular regulation during immune responses.