Phosphorylation of dynamin-related protein 1 at Ser616 regulates mitochondrial fission and is involved in mitochondrial calcium uniporter-mediated neutrophil polarization and chemotaxis

Highlights

• MCU promotes neutrophil polarization and chemotaxis.

• MCU regulates F-actin accumulation at the uropod of neutrophils during polarization.

• MCU mediates mitochondrial Ca²⁺ uptake in neutrophils.

• MCU is responsible for Drp1 (S616)-dependent mitochondrial fission in neutrophils.

• Suppression of Drp1 inhibits neutrophil polarization and chemotaxis.

Abstract

During an inflammatory response, polarization of neutrophils is necessary for effective chemotaxis and bacterial endocytosis. Ca²⁺ uptake into mitochondria through the mitochondrial calcium uniporter (MCU) is crucial for cell metabolism, signaling and survival; however, the physiological role of MCU in human neutrophils remains unclear. Here we show that MCU is vital for the polarization and chemotaxis of neutrophils. Activation of MCU by spermine promotes neutrophil polarization and chemotaxis, whereas inhibition of MCU by Ru360 blunts both processes. We also provide evidence that this role of the MCU in neutrophils may result from modulation of mitochondrial fission by increased levels of pDrp1 S616 via accumulation of Ca²⁺ into the mitochondrial matrix. Thus, our study identifies the dependence of neutrophil polarization and chemotaxis on the MCU and highlights the importance of regulating mitochondrial fission during the anti-inflammatory cascade in human neutrophils.

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.