Phosphorylation of dynamin-related protein 1 at Ser616 regulates mitochondrial fission and is involved in mitochondrial calcium uniporter-mediated neutrophil polarization and chemotaxis
Introduction
Neutrophils are the most abundant circulating leukocytes and the fastest moving cells in humans. They are key players in innate immunity and are rapidly recruited to infection and injury sites (Mollinedo et al., 1999, Nathan, 2006). At the very start of the process, neutrophils undergo asymmetric changes in cell morphology (polarization) and migrate (chemotaxis) in response to an extracellular directional chemoattractant (Lauffenburger and Horwitz, 1996, Weiner et al., 1999). Neutrophils can be activated by a variety of chemoattractants, such as complement fragment C5a, formylated peptides like N-formyl-Met-Leu-Phe (fMLP), platelet activating factor and interleukin 8 (IL-8) (Bickel, 1993, Henson, 1981, Shuster et al., 1997, Zhang et al., 1995). Of these, fMLP is a prototypic chemotactic factor (Seely et al., 2003). To achieve polarization and chemotaxis, neutrophils organize a defined anterior (leading edge) and posterior (uropod) to form spatial and functional asymmetry (Lauffenburger and Horwitz, 1996, Sanchez-Madrid and Del, 1999). However, abnormal activation of neutrophils may result in autoimmune diseases (Kaplan, 2013). Therefore, elucidating the mechanisms regulating neutrophil polarization and chemotaxis could potentially lead to novel therapeutic strategies for counteracting chronic activation of neutrophils which can cause tissue damage.
The mitochondrial calcium uniporter (MCU) channel is responsible for Ru360 and spermine-sensitive mitochondrial calcium uptake (Kirichok et al., 2004, Zhang et al., 2006). In 2011, Rizzuto and Mootha identified the key protein of the MCU, known as CCDC109A, as necessary and sufficient for mitochondrial Ca2+ uptake (Baughman et al., 2011, De Stefani et al., 2011). Later, investigations affirmed that the MCU was a multimer and the components have since been identified. They coordinately regulate mitochondrial Ca2+ uptake to balance mitochondrial function (De Stefani et al., 2015). However, how the MCU contributes to neutrophil polarization and chemotaxis remains unclear.
Dynamin-related protein 1 (Drp1) is one of the major regulatory proteins required to maintain mitochondrial morphology, maintaining the balance between continuous fusion and fission (Chen and Chan, 2004, Praefcke and McMahon, 2004). Drp1 requires activation by multiple post-translation modifications and receptor interactions before this GTPase can regulate mitochondrial fission (Cho et al., 2013). Phosphorylation of serine 637 (S637) of human Drp1 suppresses mitochondrial translocation and GTPase activity (Chang and Blackstone, 2007, Cribbs and Strack, 2007). However, phosphorylation at serine 616 (S616) enhances the activity of Drp1 and results in mitochondrial fragmentation (Cho et al., 2013). As a selective chemical inhibitor of Drp1, mdivi-1 disrupts the mitochondrial network by blunting mitochondrial fusion and fission (Park et al., 2011). Recent reports have suggested that mitochondrial fission mediated by the MCU may occur in the cerebral cortexes of rats and in the hippocampus during ischemia/reperfusion injury (Liang et al., 2014, Zhao et al., 2015).
It remains unknown whether mitochondrial fission is affected by the MCU in neutrophils and whether mitochondrial fission participates in human neutrophil polarization and chemotaxis. We incubated purified human neutrophils with spermine and Ru360 and observed changes to neutrophil motility and mitochondrial morphological alterations. We also used mdivi-1 to inhibit Drp1 to further investigate the hypothesis.
Section snippets
Reagents and antibodies
Spermine (S3256), Mdivi-1 (M0199) and fMLP (F3506) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ru360 (557440) was purchased from Calbiochem (Bad Soden, Germany). IL-8 was purchased from PeproTech (London, UK). Phosphate-buffered saline (PBS), Hank's buffered salt solution (HBSS) and d-Hank’s buffered salt solution (dHBSS) were purchased from Genom (Hangzhou, China). Dextran T500 was purchased from Amersham Biosciences (Piscataway, NJ, USA). Rhodamine phalloidin (R415), Fluo 4-AM
MCU activation promotes neutrophil polarization and chemotaxis, whereas MCU inhibition blunts both processes
To investigate the effect of the MCU on neutrophil polarization and chemotaxis, we used Zigmond chambers and transwell inserts for fMLP or IL-8 induction. Purified human neutrophils were pretreated with or without spermine or Ru360 for 30 min prior to induction with fMLP or IL-8 at different concentrations. As shown in Fig. 1A and B, neutrophils preincubated with 20 μM spermine exhibited significantly greater polarized morphology induced by 100 nM fMLP than neutrophils preincubated with control
Discussion
Neutrophils play a crucial role in the immune response and are often the first responders to inflammatory stimuli (Mollinedo et al., 1999, Nathan, 2006). Upon sensing inflammation, they polarize and migrate rapidly, regulated by the spatially restricted recruitment and activation of signaling molecules (Iijima et al., 2002, Postma et al., 2004, Van Haastert and Devreotes, 2004). Despite recent progress, the molecular mechanisms mediating neutrophil polarization and chemotaxis in the
Conflict of interests
The authors declare that they have no conflicts of interests.
Acknowledgements
This work was supported by funding from the National Natural Science Foundation of China (81502799) and the Natural Science Foundation of Guangdong Province (2015A030310100). We would like to thank Dr. Jiabao Xu at Southern Medical University, School of Public Health for reviewing and revising this paper.
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