A6: PKD-mediated regulation of the subcortical actin cytoskeleton during trauma-induced extravasation of neutrophil granulocytes

PIs: T. Seufferlein, S. Paschke

Neutrophil granulocytes are an essential element of the early inflammatory response to mechanical tissue trauma. The extravasation of neutrophils represents a crucial checkpoint during activation of the innate immune system. Thus, this process is considered an important pharmacological target for the modulation of inflammation. However, the understanding of the molecular processes controlling the passage of neutrophils from the endothelial layer into spatially confined tissue channels is incomplete. Extravasation requires neutrophils to modulate cell shape and mechanics. The actin filament network is a major compartment of the cytoskeleton in neutrophils and plays an important part in controlling these processes. Importantly, modulators of actin network architecture were found to be significantly upregulated within the first 12 hours after tissue trauma. Protein Kinases D (PKD) family as serine/threonine kinases are crucial regulators during the dynamic rearrangement of actin filaments. Myeloid restricted inactivation of PKD1 has recently been shown to affect neutrophil migration and cause exacerbations of bacterial chest infections. However, the mechanisms of this regulation in particular at the level of the actin cytoskeleton remain to be elucidated.

In this project we will investigate the role of PKD family members for modulating the architecture of the actin cytoskeleton and, thereby, motility of neutrophils in response to mechanical trauma. In particular, we plan to study how blunt chest trauma induces specific activation patterns of PKD isoforms in neutrophils. To examine the impact of PKD activation on cellular mechanics and motility of primary neutrophils, we will use a wide range of innovative techniques, in particular optical stretching for investigating cell mechanics and microfluidic migration channels to study cell motility in spatial confinement. Actin network architecture will be studied by scanning electron microscopy. Neutrophil-like NB4 cells will be used to characterise actin-regulatory PKD substrates and investigate interactions between PKD and the actin cytoskeleton by interfering with PKD-associated signalling pathways. The results will help to define potential targets to modulate posttraumatic inflammation by addressing the regulation of the actin cytoskeleton.

Video

Projektleiter

Prof. Dr. Thomas Seufferlein
Zentrum für Innere Medizin
Klinik für Innere Medizin I
Albert-Einstein-Allee 23
89081 Ulm
Tel.: +49 731 500 44501
Fax: +49 731 500 44502
thomas.seufferlein(at)uniklinik-ulm.de
Homepage

Dr. med. Stephan Paschke
Zentrum für Chirurgie
Klinik für Allgemein- und Viszeralchirurgie
Albert-Einstein-Allee 23
89081 Ulm
Tel.: +49 731 500 53596
Fax: +49 731 500 53502
stephan.paschke(at)uniklinik-ulm.de
Homepage

Extravasation (A) and interstitial movement (B) of neutrophils. Both aspects of neutrophil migration require highly dynamic shape changes that require a considerable amount of deformability. The actin filament network is a major compartment of the cytoskeleton in neutrophils and plays an important part in controlling these processes. Protein Kinases D (PKD) family are crucial regulators during the dynamic rearrangement of actin filaments.
Visualisation of the subcortical cytoskeleton by scanning electron microscopy. Inset (A) depicts a human neutrophil. The cell membrane (m) was partially removed to reveal the filamentous cytoskeleton (c) within the subcortical compartment. The filaments are composed of actin as demonstrated in inset (B) by immunogold labelling with an anti-actin antibody (red dots). The scale bars represent 150 nm.