Neutrophils Establish Rapid and Robust WAVE Complex Polarity in an Actin-Dependent Fashion

Abstract

Asymmetric intracellular signals enable cells to migrate in response to external cues. The multiprotein WAVE (SCAR/WASF) complex activates the actin-nucleating Arp2/3 complex [1-4] and localizes to propagating “waves”, which direct actin assembly during neutrophil migration [5, 6]. Here, we observe similar WAVE complex dynamics in other mammalian cells and analyze WAVE complex dynamics during the establishment of neutrophil polarity. Earlier models proposed that either spatially-biased generation [7] or selection of protrusions [8] enables chemotaxis. These models require existing morphological polarity to control protrusions. Similar spatially-biased generation and selection of WAVE complex recruitment occur in morphologically unpolarized neutrophils during the development of their first protrusions. Additionally, several mechanisms limit WAVE complex recruitment during polarization and movement: intrinsic cues restrict WAVE complex distribution during the establishment of polarity, and asymmetric intracellular signals constrain WAVE complex distribution in morphologically polarized cells. External gradients can overcome both intrinsic biases and control WAVE complex localization. Following latrunculin-mediated inhibition of actin polymerization, addition and removal of agonist gradients globally recruits and releases the WAVE complex from the membrane. Under these conditions the WAVE complex no longer polarizes, despite the presence of strong external gradients. Thus, actin polymer and the WAVE complex reciprocally interact during polarization.

The Endoplasmic Reticulum Is a Reservoir for WAVE/SCAR Regulatory Complex Signaling in the Arabidopsis Leaf

“…In addition, biochemical studies that employ soluble vertebrate WAVE/SCAR proteins or fully assembled W/SRC detect specific binding to phospholipids that is consistent with a cytosolic recruitment model (Oikawa et al, 2004;Steffen et al, 2004;Lebensohn and Kirschner, 2009). However, in many motile cell types, including neurons, NAP1 has a striking punctate or perinuclear localization pattern that may reflect the general importance of multiple organelles in the cellular cycles of W/SRC activation and use (Bogdan and Klämbt, 2003;Steffen et al, 2004;Millius et al, 2009).…”

Diffusion, capture and recycling of SCAR/WAVE and Arp2/3 complexes observed in cells by single-molecule imaging

“…Furthermore, actin polymer helps to sculpt the spatial distribution of the WAVE complex in response to external stimuli (Millius et al, 2009). However, these experiments relied on treating cells with single pharmacological inhibitors of actin assembly such as the actin-sequestering drug latrunculin.…”

Actin dynamics rapidly reset chemoattractant receptor sensitivity following adaptation in neutrophils

“…Glass capillaries were pulled as described [7] except that they were pulled on a Sutter Model P-97 instead of P-87 (exact program varies based on heating filament properties). Needles were backfilled with a sterile-filtered solution (0.2 mm pore size) containing 1 mM fMLP (F3506, Sigma) and 1 mM Alexa 594 sodium hydrazide (A-10438, Invitrogen) and held by a micromanipulator (MM-89, Narishige).…”

“…Needles were backfilled with a sterile-filtered solution (0.2 mm pore size) containing 1 mM fMLP (F3506, Sigma) and 1 mM Alexa 594 sodium hydrazide (A-10438, Invitrogen) and held by a micromanipulator (MM-89, Narishige). Agonist flow rate from the pipette was controlled as described [7].…”

“…Custom scripts using the MATLAB image processing toolbox were written to define cell edge, and heatmaps showing PI3K lipid product intensity across the cell edge were generated as described [7]. Position of micropipette was also calculated as described [7]. For calcium assays, raw data was preprocessed in SOFTMAX PRO v. 5.4 (as described in the electronic supplementary material, figure S1) and transferred to an Excel spreadsheet for plotting.…”