Short microvilli cover the surfaces of circulating mammalian lymphocytes. The surfaces of monocytes and neutrophils are very different, containing ruffles as their predominant structure. In this study, we present the first quantitative characterization of lymphocyte microvilli. From analysis of scanning electron micrographs, we find that median microvillar length and surface density range from 0.3 to 0.4 m and 2 to 4 microvilli/m 2 , respectively, on lymphocytes from a variety of sources. As with similar structures from other cells, lymphocyte microvilli contain parallel bundles of actin filaments. Lymphocyte microvilli rapidly disassemble when exposed to the actin-sequestering molecule, Latrunculin A. This disassembly parallels cellular actin filament depolymerization and is complete within 2 minutes, suggesting that lymphocyte microvilli undergo continuous assembly and disassembly. In contrast to previous reports suggesting lymphocyte microvillar density to be reduced on lymphocytes from Wiskott-Aldrich syndrome (WAS) patient, we find no such deficiency in either mouse or human WAS protein (WASp)-deficient lymphocytes. These results suggest that WASp is either not involved in or is redundant in the rapid dynamics of lymphocyte microvilli. IntroductionCell surfaces are rarely flat and generally contain a variety of protrusions and/or invaginations. Microvilli are fingerlike protrusive structures found on the surfaces of many cells and are abundant on the surfaces of circulating T and B lymphocytes. 1 While definitive roles for lymphocyte microvilli have not been established, a proposed function is in segregation of surface receptors during extravasation. 2 Two receptors involved in the initial rolling phase of extravasation, L-selectin 3,4 and ␣4␤7 integrin, 5 localize to microvillar tips. In contrast, the ␤2 integrins that mediate subsequent stable adhesion and diapedesis localize to nonprotrusive regions of the cell surface. 5 This spatial segregation of surface receptors might enable a temporal segregation of adhesive function during extravasation. Lymphocytes expressing chimeric L-selectin constructs that no longer localize to microvilli do not roll on L-selectin ligands, 3 supporting this hypothesis. Microvilli are correlated with metastatic potential of certain cancers, 6 perhaps due to their role in extravasation. In addition, receptors for HIV, including CD4, CC chemokine receptor 5, and CXC chemokine receptor 4, localize to microvilli, 7 suggesting a potential role for microvilli in HIV infection.Other fingerlike protrusions on cell surfaces, including epithelial microvilli and surface-attached filopodia on many cell types, depend on actin filaments for their structure. 8,9 These filaments are organized in parallel bundles that appear to span the length of the microvillus and are cross-linked to each other and to the microvillar membrane by a number of proteins. 10 The dynamics of these structures vary immensely. While filopodia grow and shrink intermittently by alternate assembly and disassembly of t...

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.

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.