Actin-based protrusions of migrating neutrophils are intrinsically lamellar and facilitate direction changes.

Leukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods of cells moving in three dimensions. We therefore employed lattice light sheet microscopy to perform three-dimensional, time-lapse imaging of neutrophil-like HL-60 cells crawling through collagen matrices. To analyze three-dimensional pseudopods we: (i) developed fluorescent probe combinations that distinguish cortical actin from dynamic, pseudopod-forming actin networks, and (ii) adapted molecular visualization tools from structural biology to render and analyze complex cell surfaces. Surprisingly, three-dimensional pseudopods turn out to be composed of thin (<0.75 µm), flat sheets that sometimes interleave to form rosettes. Their laminar nature is not templated by an external surface, but likely reflects a linear arrangement of regulatory molecules. Although we find that Arp2/3-dependent pseudopods are dispensable for three-dimensional locomotion, their elimination dramatically decreases the frequency of cell turning, and pseudopod dynamics increase when cells change direction, highlighting the important role pseudopods play in pathfinding.

Arp2 depletion inhibits sheet-like protrusions but not linear protrusions of fibroblasts and lymphocytes.

The Arp2/3 complex-mediated assembly and protrusion of a branched actin network at the leading edge occurs during cell migration, although some studies suggest it is not essential. In order to test the role of Arp2/3 complex in leading edge protrusion, Swiss 3T3 fibroblasts and Jurkat T cells were depleted of Arp2 and evaluated for defects in cell morphology and spreading efficiency. Arp2-depleted fibroblasts exhibit severe defects in formation of sheet-like protrusions at early time points of cell spreading, with sheet-like protrusions limited to regions along the length of linear protrusions. However, Arp2-depleted cells are able to spread fully after extended times. Similarly, Arp2-depleted Jurkat T lymphocytes exhibit defects in spreading on anti-CD3. Interphase Jurkats in suspension are covered with large ruffle structures, whereas mitotic Jurkats are covered by finger-like linear protrusions. Arp2-depleted Jurkats exhibit defects in ruffle assembly but not in assembly of mitotic linear protrusions. Similarly, Arp2-depletion has no effect on the highly dynamic linear protrusion of another suspended lymphocyte line. We conclude that Arp2/3 complex plays a significant role in assembly of sheet-like protrusions, especially during early stages of cell spreading, but is not required for assembly of a variety of linear actin-based protrusions.