Directional Migration and Cytoskeletal Organization in Human Neutrophils

Directional migration and cytoskeletal organization in human neutrophils

Branden T. Kusanto,¹ Asako Itakura,² Robert H. Insall,³ Owen J. T. McCarty^1,2,4

¹Department of Biomedical Engineering, ^­2Department of Cell & Developmental Biology, ⁴Division of Hematology / Medical Oncology, School of Medicine, Oregon Health & Science University, Portland, OR, USA; ³CRUK Beatson Institute for Cancer Research, Glasgow, United Kingdom; 

 

Neutrophils play a key role in human physiology by acting as the first line of defense within the immune system. These white blood cells travel towards the sign of infection using a process called chemotaxis. Chemotaxis is comprised of three steps, directional sensing, polarization and motility. In the first step, directional sensing, neutrophils constantly look for the signs of infection and orient themselves to the site of infection. The second step, polarization, causes the neutrophil to have a leading edge and tail, which allows the neutrophil to migrate. The last step of neutrophil migration is motility; it is mediated by actin cytoskeletal reorganzation. The molecular mechanisms underlying these dynamic processes are still poorly understood.

At the site of infection, bacteria release a chemoattractant f-Met-Leu-Phe (fMLP). Neutrophils recognize the chemoattractant through fMLP receptors. This signal proceeds through downstream pathways activating phosphoinositide 3-kinases (PI3Ks) and Rac. In turn, Rac then activates p21-activated kinases (PAKs). PAK activation has been shown to regulate the processes of cell morphology, polarity, motility, division, macropinocytosis (cathrin-independent), and invasiveness.  

This study was aimed to determine the signaling mechanisms that regulate the processes of neutrophil chemotaxis. Purified neutrophils were treated with different inhibitors that specifically target signaling components of the Rac-PAK axis, such as Rac, PAK, PI3K, and Rho-associated protein kinase (ROCK). Neutrophil migration was characterized using live-cell video microscopy under a gradient of fMLP. ImageJ was used to track the migrating cells. I characterized the dynamic profile of neutrophil chemotaxis using a MatLab program to create spider plots, rose plots, and velocity v. time graphs. In parallel, I used immunostaining techniques to characterized protein localization in fMLP-stimulated neutrophils at a molecular level in order to localize the spatial distribution of PAK in migrating neutrophils for the first time.

Under an fMLP gradient, our results show that neutrophils undergo chemotaxis with distinct leading edges and tails, migrating toward the fMLP gradient with a speed of 7.5 ± 0.56 mm/min. Rac, Our results show that pharmacologic inhibitors of PI3k and ROCK impaired directionality, yet did not affect the migration speed of neutrophils. Moreover, neutrophils treated with PAK inhibitor not only demonstrated random migration but also exhibited less polarization and reduced motility. Our results obtained by immunostaining show that human neutrophils express PAK 1, 2, and 4. Upon the stimulation with fMLP, PAK2 was localized at actin-rich leading edge. Taken together, this study indicates PAK2 may play a key role in regulating directionality and motility during neutrophil chemotaxis.