Abstract
BackgroundMigrating leukocytes normally have a polarized morphology with an actin-rich lamellipodium at the front and a uropod at the rear. Microtubules (MTs) are required for persistent migration and chemotaxis, but how they affect cell polarity is not known.Methodology/Principal FindingsHere we report that T cells treated with nocodazole to disrupt MTs are unable to form a stable uropod or lamellipodium, and instead often move by membrane blebbing with reduced migratory persistence. However, uropod-localized receptors and ezrin/radixin/moesin proteins still cluster in nocodazole-treated cells, indicating that MTs are required specifically for uropod stability. Nocodazole stimulates RhoA activity, and inhibition of the RhoA target ROCK allows nocodazole-treated cells to re-establish lamellipodia and uropods and persistent migratory polarity. ROCK inhibition decreases nocodazole-induced membrane blebbing and stabilizes MTs. The myosin inhibitor blebbistatin also stabilizes MTs, indicating that RhoA/ROCK act through myosin II to destabilize MTs.Conclusions/SignificanceOur results indicate that RhoA/ROCK signaling normally contributes to migration by affecting both actomyosin contractility and MT stability. We propose that regulation of MT stability and RhoA/ROCK activity is a mechanism to alter T-cell migratory behavior from lamellipodium-based persistent migration to bleb-based migration with frequent turning.
Highlights
Cell migration is essential for the recruitment of T cells to and circulation within lymphoid organs, where they encounter antigen-presenting dendritic cells, and in tissues during immune surveillance, immune responses and inflammation
We propose that regulation of MT stability and RhoA/ROCK activity is a mechanism to alter T-cell migratory behavior from lamellipodium-based persistent migration to bleb-based migration with frequent turning
Lamellipodium extension in T cells requires Rac-induced actin polymerization [13], whereas the uropod is enriched in cell adhesion molecules such as ICAM-3 and CD44 that associate with ezrin/radixin/moesin (ERM) proteins, which in turn link these receptors with the cortical actin cytoskeleton [14]
Summary
Cell migration is essential for the recruitment of T cells to and circulation within lymphoid organs, where they encounter antigen-presenting dendritic cells, and in tissues during immune surveillance, immune responses and inflammation. Migrating T cells are normally morphologically polarized with spatially distinct front (lamellipodium) and rear (uropod) structures, and migrate by extending the lamellipodium forwards and retracting the uropod [1,2,3]. Cell polarization and migration require dynamic rearrangement of the actin and microtubule cytoskeletons via intracellular signaling pathways involving Rho family GTPases [10,11,12]. Rho signaling is required for uropod extension and for detachment of the rear of migrating T cells [3,15]. Migrating leukocytes normally have a polarized morphology with an actin-rich lamellipodium at the front and a uropod at the rear. Microtubules (MTs) are required for persistent migration and chemotaxis, but how they affect cell polarity is not known
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