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Rac is required for constitutive macropinocytosis by dendritic cells but does not control its downregulation
M A West1, A R Prescott, E L Eskelinen
1Department of Biochemistry, MSI/WTB Complex, University of Dundee, Dundee, DDI 5EH, UK. c.watts@dundee.ac.uk
Background:
Dendritic cells use constitutive macropinocytosis to capture exogenous antigens for presentation on MHC molecules. Upon exposure to inflammatory stimuli or bacterial products such as lipopolysaccharide (LPS), macropinocytosis is dramatically downregulated as part of a developmental programme leading to dendritic cell maturation, migration and activation of T cells. It is not known, however, how macropinocytosis is sustained in dendritic cells in the absence of exogenous stimuli, nor how it is downregulated upon maturation. We have tested the possibility that one or more members of the Rho family of GTPases are involved in and control pinocytosis in dendritic cells.
Results:
We established dendritic cell populations that show constitutive macropinocytosis that was downregulated by LPS treatment. Microinjection of immature cells with dominant-negative Rac (N17Rac1) or treatment with Clostridium difficile toxin B, the phosphoinositide 3-kinase (PI3-K) inhibitor wortmannin, or LPS all inhibited the formation of macropinosomes but, surprisingly, did not eliminate membrane ruffling. Microinjection of N17Cdc42 or the Rho inhibitor C3 transferase eliminated actin plaques/podosomes and actin cables, respectively, but had little effect on the formation of macropinosomes. Surprisingly, dendritic cells matured with LPS had equivalent or even somewhat higher levels of active Rac than immature cells. Moreover, microinjection of a constitutively active form of Rac (V12Rac1) into mature dendritic cells did not reactivate macropinocytosis.
Conclusions:
Rac has an important role in the constitutive formation of macropinosomes in dendritic cells but may be required downstream of membrane ruffling. Furthermore, regulation of Rac activity does not appear to be the control point in the physiological downregulation of dendritic cell pinocytosis. Instead, one or more downstream effectors may be modulated to allow Rac to continue to regulate other cellular functions.
Insights
Rac regulates constitutive macropinocytosis in dendritic cells but is not the primary control for its downregulation during maturation. Downstream effectors, not Rac activity, likely mediate this process.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Dendritic cells (DCs) constitutively use macropinocytosis to internalize antigens for T cell presentation.
- Macropinocytosis is downregulated during DC maturation induced by inflammatory stimuli like lipopolysaccharide (LPS).
- Mechanisms sustaining basal macropinocytosis and its downregulation during maturation remain unclear.
Purpose of the Study:
- Investigate the role of Rho family GTPases in regulating macropinocytosis in dendritic cells.
- Determine how macropinocytosis is controlled and downregulated during dendritic cell maturation.
Main Methods:
- Established dendritic cell populations exhibiting constitutive macropinocytosis.
- Utilized dominant-negative Rac (N17Rac1), Clostridium difficile toxin B, PI3-K inhibitor wortmannin, LPS, N17Cdc42, and C3 transferase.
- Assessed effects on macropinosome formation, membrane ruffling, actin plaques/podosomes, and actin cables.
Main Results:
- Inhibition of Rac, PI3-K, or LPS treatment reduced macropinosome formation but not membrane ruffling.
- Cdc42 inhibition affected actin structures but not macropinosome formation; Rho inhibition affected actin cables.
- Mature DCs showed similar or higher active Rac levels than immature DCs; active Rac did not restore macropinocytosis in mature DCs.
Conclusions:
- Rac is crucial for constitutive macropinosome formation in DCs, acting downstream of membrane ruffling.
- Rac activity regulation is not the key control point for the physiological downregulation of DC pinocytosis.
- Downstream effectors of Rac, rather than Rac activity itself, are likely modulated during DC maturation to control pinocytosis.