Abstract
ABSTRACTSustained forward migration through a fibrillar extracellular matrix requires localization of protrusive signals. Contact with fibronectin at the tip of a cell protrusion activates Rac1, and for linear migration it is necessary to dampen Rac1 activity in off-axial positions and redistribute Rac1 from non-protrusive membrane to the leading edge. Here, we identify interactions between coronin-1C (Coro1C), RCC2 and Rac1 that focus active Rac1 to a single protrusion. Coro1C mediates release of inactive Rac1 from non-protrusive membrane and is necessary for Rac1 redistribution to a protrusive tip and fibronectin-dependent Rac1 activation. The second component, RCC2, attenuates Rac1 activation outside the protrusive tip by binding to the Rac1 switch regions and competitively inhibiting GEF action, thus preventing off-axial protrusion. Depletion of Coro1C or RCC2 by RNA interference causes loss of cell polarity that results in shunting migration in 1D or 3D culture systems. Furthermore, morpholinos against Coro1C or RCC2, or mutation of any of the binding sites in the Rac1–RCC2–Coro1C complex delays the arrival of neural crest derivatives at the correct location in developing zebrafish, demonstrating the crucial role in migration guidance in vivo.
Highlights
The persistence and direction of in vivo cell migration is essential during development and wound healing, meaning that localization and turnover of protrusive signals are crucial
RCC2 as a non-canonical Rac1-sequestering molecule Persistent migration requires polarization of signals, such as Rac1 and, it is known that concerted engagement of fibronectin receptors, a5b1 integrin and syndecan-4, triggers Rac1 activation (Bass et al, 2007), it is less clear how the signal remains localized to a single protrusion as the composition of a 3D matrix changes
RCC2 has been identified as a negative regulator of Rac1 that associates with adhesion complexes (Humphries et al, 2009), leading us to investigate the role of a putative Rac1 inhibitor in a signaling complex traditionally linked to Rac1 activation
Summary
The persistence and direction of in vivo cell migration is essential during development and wound healing, meaning that localization and turnover of protrusive signals are crucial. Defective Rac signaling in Danio rerio (zebrafish) upon injection of morpholinos against the receptor responsible for Rac activation, syndecan-4 (Bass et al, 2007), causes More recently, other putative Rac regulators have been identified that might provide the spatial and temporal resolution required in vivo One such factor is RCC2 which, despite being initially characterized for its role in mitotic spindle assembly (Mollinari et al, 2003), was recently found to be a component of fibronectin-associated adhesion complexes (Humphries et al, 2009). By associating with the actin cytoskeleton or adhesion complexes, RCC2 and coronins are suitably localized to regulate GTPases and the key question is whether they act as non-canonical GEFs, GAPs or GDIs, second regulators of existing mechanisms or influence GTPases by modulating their localization
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