Three hundred years, ago Anton van Leeuwenhoek’s simple microscope revealed ‘animacules’ changing shape as they moved through a drop of water (van Leewenhoeck, 1677). Subsequent breakthroughs in microscope technology and molecular biology have strengthened the link between cell morphology and locomotion, and we now know that crawling cells from many eukaryotic phyla create a variety of dynamic protrusions that project forward, in the direction of migration.

Thin, flat protrusions produced by strongly adherent cells are generally called lamellipodia. Previous work, mainly on fibroblasts and epithelial cells, has established that movement of leading-edge lamellipodia is driven by growth of branched actin networks nucleated and crosslinked by the Arp2/3 complex. As they advance, these lamellipodia promote creation of new focal adhesions that provide traction and support continued movement (Case and Waterman, 2015). This mode of cell migration appears to be restricted to the animal lineage, where it relies on the presence of specific ligands in the extracellular environment and plays essential roles in embryonic development, wound healing, and tissue homeostasis.

A more widely dispersed form of cell crawling—probably present in the last common ancestor of all eukaryotes (Fritz-Laylin et al., 2017)—requires only weak, non-specific interaction with the extracellular environment and can achieve speeds several orders of magnitude faster than adhesion-based movement (Loomis et al., 2012; Preston and King, 1978; Lämmermann et al., 2008; Buenemann et al., 2010; Butler et al., 2010; Loomis et al., 2012; Petrie and Yamada, 2015; Fritz-Laylin et al., 2017). This mode of cell migration is associated with formation of complex, three-dimensional pseudopods filled with branched networks of actin filaments, nucleated and organized by the Arp2/3 complex. These three-dimensional pseudopods may contribute to forward motion of crawling and swimming cells (Van Haastert, 2011), but recent work has revealed that disrupting pseudopod formation can increase the speed and persistence of migration (Leithner et al., 2016; Vargas et al., 2016), implying that complex pseudopods might be more important for exploring the external environment than for driving forward motion.

We know less about the morphology and function of complex pseudopods than adherent lamellipodia in part due to the technical limitations of live-cell light microscopy. Confocal and total internal reflection fluorescence (TIRF) microscopy, for example, provide adequate views of adherent lamellipodia because these structures are: (i) slow-moving, (ii) thin, and (iii) closely adhered to the microscope coverslip. In contrast, the complex pseudopods created by fast-moving cells: (i) grow quickly (10–100 um/min), (ii) adopt complex three-dimensional shapes, and (iii) often spend their lives far from a coverslip surface. In addition, photobleaching must be minimized in order to observe living cells for sufficient time to track their complex three-dimensional migration.

Although some work has suggested that two-dimensional lamellipodia of adherent cells reflect the flatness of the surface to which they are attached (Burnette et al., 2014), no mechanisms have been proposed to explain the more complex morphologies of three-dimensional pseudopods created by fast, weakly adherent cells—especially when crawling through irregular environments. Moreover, it remains unclear whether these complex pseudopods are completely amorphous, or whether they share common, underlying structural features that might shed light on their functions and mechanisms of assembly.

To address these issues, we employed the recently developed lattice light sheet microscope (Chen et al., 2014), which offers a unique combination of three-dimensional imaging with high spatial and temporal resolution and low phototoxicity. To render and interpret the large data sets produced by lattice light sheet microscopy, we developed new visualization software and combined it with biochemically defined molecular probes to detect and characterize pseudopods in living cells.

Using these new tools, we discovered that pseudopods created by fast-moving neutrophils are not entirely amorphous, but represent various arrangements of a single structural motif: a free-standing lamellar sheet. Thus, complex, three-dimensional pseudopods that appear amorphous in widefield and confocal microscopy turn out to be rosettes formed of multiple, interleaved lamella. Unlike adherent lamellipodia, these free-standing lamellar building blocks are not templated from a flat surface and their morphology strongly suggests that they arise from a linear arrangement of regulatory molecules associated with the plasma membrane. In addition, our automated methods for detecting and quantifying the size of complex pseudopods reveal that these dynamic cellular structures play a key role in cellular pathfinding.