The role of adenomatous polyposis coli (APC)-mediated actin assembly in microtubule-capture and focal adhesion turnover

Focal adhesions (FA) are highly dynamic structures that assemble at the front of the cell
and disassemble at the rear, allowing cells to move directionally. Focal adhesion (FA)
turnover depends on microtubules and actin. Microtubule ends are captured at FAs,
where they induce rapid FA disassembly. However, actin’s roles are less clear. Here, we use
polarization-resolved microscopy, FRAP, super-resolution microscopy, live-cell imaging,
and a mutant of Adenomatous polyposis coli that is incapable of nucleating actin
(APC-m4) and impairs directional cell migration (Juanes et al., 2017; J. Cell Biology) to
investigate the role of actin assembly in FA turnover. We show that APC-mediated actin
assembly is critical for maintaining normal F-actin levels, organization, and dynamics at
FAs, along with proper organization of FA components, including Src and FAK Kinases,
and Paxillin. Live imaging in wild type cells shows that microtubules are captured
repeatedly at FAs as they mature, but once a FA reaches peak maturity, the next
microtubule capture event leads to delivery of an autophagosome, triggering FA
disassembly. In APC-m4 cells, microtubule capture frequency and duration are altered,
and there are long delays between autophagosome delivery and FA disassembly. Thus,
APC-mediated actin assembly is required for normal feedback between microtubules and
FAs, and maintaining FAs in a state ‘primed’ for microtubule-induced turnover. In addition,
we found that APC-m4 expression or silencing of the formin Dia1 each caused a ~30%
reduction in total F-actin levels in cells; however, Dia1 silencing led to only modest defects
in FA turnover. Thus, APC-m4 effects on FA turnover do not arise from a general loss of
actin assembly in cells, but rather from a specific disruption of APC-mediated actin
nucleation at FAs. In summary, this work demonstrates that APC is a bona fide actin
nucleator in vivo, and that this activity of APC is crucial for regulating FA turnover
underlying directed cell migration. Importantly, our results do not rule out the possibility
of other actin assembly-promoting factors contributing to FA turnover, or APC
coordinating actin and microtubule dynamics in other cellular locations besides FAs