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Contact guidance persists under myosin inhibition due to the local alignment of adhesions and individual protrusions.

ABSTRACT: Contact guidance-cell polarization by anisotropic substrate features-is integral to numerous physiological processes; however the complexities of its regulation are only beginning to be discovered. In particular, cells polarize to anisotropic features under non-muscle myosin II (MII) inhibition, despite MII ordinarily being essential for polarized cell migration. Here, we investigate the ability of cells to sense and respond to fiber alignment in the absence of MII activity. We find that contact guidance is determined at the level of individual protrusions, which are individually guided by local fiber orientation, independent of MII. Protrusion stability and persistence are functions of adhesion lifetime, which depends on fiber orientation. Under MII inhibition, adhesion lifetime no longer depends on fiber orientation; however the ability of protrusions to form closely spaced adhesions sequentially without having to skip over gaps in adhesive area, biases protrusion formation along fibers. The co-alignment of multiple protrusions polarizes the entire cell; if the fibers are not aligned, contact guidance of individual protrusions still occurs, but does not produce overall cell polarization. These results describe how aligned features polarize a cell independently of MII and demonstrate how cellular contact guidance is built on the local alignment of adhesions and individual protrusions.

SUBMITTER: Kubow KE

PROVIDER: S-EPMC5662575 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Contact guidance persists under myosin inhibition due to the local alignment of adhesions and individual protrusions.

Kubow Kristopher E KE Shuklis Victoria D VD Sales Dominic J DJ Horwitz A Rick AR

Scientific reports 20171030 1

Contact guidance-cell polarization by anisotropic substrate features-is integral to numerous physiological processes; however the complexities of its regulation are only beginning to be discovered. In particular, cells polarize to anisotropic features under non-muscle myosin II (MII) inhibition, despite MII ordinarily being essential for polarized cell migration. Here, we investigate the ability of cells to sense and respond to fiber alignment in the absence of MII activity. We find that contact ...[more]

PMID: 29085052

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Project description:UnlabelledGrowth cones interact with the extracellular matrix (ECM) through integrin receptors at adhesion sites termed point contacts. Point contact adhesions link ECM proteins to the actin cytoskeleton through numerous adaptor and signaling proteins. One presumed function of growth cone point contacts is to restrain or "clutch" myosin-II-based filamentous actin (F-actin) retrograde flow (RF) to promote leading edge membrane protrusion. In motile non-neuronal cells, myosin-II binds and exerts force upon actin filaments at the leading edge, where clutching forces occur. However, in growth cones, it is unclear whether similar F-actin-clutching forces affect axon outgrowth and guidance. Here, we show in Xenopus spinal neurons that RF is reduced in rapidly migrating growth cones on laminin (LN) compared with non-integrin-binding poly-d-lysine (PDL). Moreover, acute stimulation with LN accelerates axon outgrowth over a time course that correlates with point contact formation and reduced RF. These results suggest that RF is restricted by the assembly of point contacts, which we show occurs locally by two-channel imaging of RF and paxillin. Further, using micropatterns of PDL and LN, we demonstrate that individual growth cones have differential RF rates while interacting with two distinct substrata. Opposing effects on RF rates were also observed in growth cones treated with chemoattractive and chemorepulsive axon guidance cues that influence point contact adhesions. Finally, we show that RF is significantly attenuated in vivo, suggesting that it is restrained by molecular clutching forces within the spinal cord. Together, our results suggest that local clutching of RF can control axon guidance on ECM proteins downstream of axon guidance cues.Significance statementHere, we correlate point contact adhesions directly with clutching of filamentous actin retrograde flow (RF), which our findings strongly suggest guides developing axons. Acute assembly of new point contact adhesions is temporally and spatially linked to attenuation of RF at sites of forward membrane protrusion. Importantly, clutching of RF is modulated by extracellular matrix (ECM) proteins and soluble axon guidance cues, suggesting that it may regulate axon guidance in vivo. Consistent with this notion, we found that RF rates of spinal neuron growth cones were slower in vivo than what was observed in vitro. Together, our study provides the best evidence that growth cone-ECM adhesions clutch RF locally to guide axons in vivo.

Dataset Information

Contact guidance persists under myosin inhibition due to the local alignment of adhesions and individual protrusions.

Publications

Contact guidance persists under myosin inhibition due to the local alignment of adhesions and individual protrusions.

Similar Datasets

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