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J. Cell Biol.,
Volume 145, Number 2, April 19, 1999 331-345
* Department of Anatomy and Structural Biology, and Using both light and high resolution electron
microscopy, we analyzed the spatial and temporal relationships between the Arp2/3 complex and the nucleation activity that is required for lamellipod extension
in mammary carcinoma cells after epidermal growth
factor stimulation. A rapid two- to fourfold increase in
filament barbed end number occurs transiently after
stimulation and remains confined almost exclusively to
the extreme outer edge of the extending lamellipod
(within 100-200 nm of the plasma membrane). This is accompanied by an increase in filament density at the
leading edge and a general decrease in filament length,
with a specific loss of long filaments. Concomitantly,
the Arp2/3 complex is recruited with a 1.5-fold increase
throughout the entire cortical filament network extending 1-1.5 µm in depth from the membrane at the leading edge. The recruitment of the Arp2/3 complex at the
membrane of the extending lamellipod indicates that
Arp2/3 may be involved in initial generation of growing
filaments. However, only a small subset of the complex
present in the cortical network colocalizes near free
barbed ends. This suggests that the 100-200-nm submembraneous compartment at the leading edge of the
extending lamellipod constitutes a special biochemical
microenvironment that favors the generation and maintenance of free barbed ends, possibly through the locally active Arp2/3 complex, severing or decreasing the
on-rate of capping protein. Our results are inconsistent
with the hypothesis suggesting uncapping is the dominant mechanism responsible for the generation of nucleation activity. However, they support the hypothesis
of an Arp2/3-mediated capture of actin oligomers that
formed close to the membrane by other mechanisms
such as severing. They also support pointed-end capping by the Arp2/3 complex, accounting for its wide distribution at the leading edge.
Analytical Imaging Facility, Albert Einstein College of Medicine, Bronx,
New York 10461
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