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

Abstract

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.

Mammalian Amoeboid Swimming is propelled by molecular and not protrusion-based paddling in Lymphocytes

“…Movie 5). While the front of migrating leukocytes usually displays lamellar shaped protrusions 32 , CK666-treated cells formed filopodia and blebs in the cell front. The effect of CK666 on swimming speed was found intermediate between blebbistatin and Latrunculin cases.…”

Molecular Visualization on the Holodeck

“…An example is lightsheet microscopy of neutrophils crawling through collagen filaments 20 imaged once per second for 3 minutes, with and without motility-affecting drugs. To examine whether the cells propel themselves by pushing or pulling on filaments, we can measure displacements of collagen in contact with cells (Figure 3).…”

Biased localization of actin binding proteins by actin filament conformation

“…Secondly, it is interesting to speculate that CH1-CH2 domains could be used to engineer probes for different structural states of f-actin for use both in vitro and in vivo. In fact, although utrnWT has been commonly used as a marker for f-actin (Burkel et al, 2007), it has also been reported to localize more preferentially to the trailing edge of migrating cells (Fritz-Laylin et al, 2017). Cterminal truncated forms of utrnWT have also been used for labelling of nuclear actin filaments (Belin et al, 2014) and direct fusions to GFP via a helical linker have been used in fluorescence polarization studies (Nakai et al, 2019).…”