Cdc42 and RhoA reveal different spatio-temporal dynamics upon local stimulation with Semaphorin-3A

Abstract

Small RhoGTPases, such as Cdc42 and RhoA, are key players in integrating external cues and intracellular signaling pathways that regulate growth cone (GC) motility. Indeed, Cdc42 is involved in actin polymerization and filopodia formation, whereas RhoA induces GC collapse and neurite retraction through actomyosin contraction. In this study we employed Förster Resonance Energy Transfer (FRET) microscopy to study the spatio-temporal dynamics of Cdc42 and RhoA in GCs in response to local Semaphorin-3A (Sema3A) stimulation obtained with lipid vesicles filled with Sema3A and positioned near the selected GC using optical tweezers. We found that Cdc42 and RhoA were activated at the leading edge of NG108-15 neuroblastoma cells during spontaneous cycles of protrusion and retraction, respectively. The release of Sema3A brought to a progressive activation of RhoA within 30 s from the stimulus in the central region of the GC that collapsed and retracted. In contrast, the same stimulation evoked waves of Cdc42 activation propagating away from the stimulated region. A more localized stimulation obtained with Sema3A coated beads placed on the GC, led to Cdc42 active waves that propagated in a retrograde manner with a mean period of 70 s, and followed by GC retraction. Therefore, Sema3A activates both Cdc42 and RhoA with a complex and different spatial-temporal dynamics.

In vivo topology converts competition for cell-matrix adhesion into directional migration

“…However, in growth cones Sema3A induces Rac1 49 and activates the Rho/ROCK/Myosin II pathway 50–53 . In addition, dynamics of small GTPases upon Sema3A are extremely complex in growth cones exemplified by the dynamics of waves of Cdc42 and RhoA 54 . Further, adhesion of growth cone to the substrate and the turnover of focal contacts are extremely sensitive to variation of Rac1 and RhoA levels 50 .…”

Actin dynamics in cell migration

“…Their spatiotemporal activities have been extensively studied using fluorescence resonance energy transfer microscopy, with both being active at leading edge and cell rear, in principle, although with differential patterns and magnitudes [40–42]. Although not absent in protrusion, which still constitutes a matter of vivid debate [42–45], it appears that RhoA signaling is at least enhanced at retraction sites [40,41]. Indeed, there is mutual inhibition at multiple levels between Rac1 and RhoA signaling, which can lead to a bistable system [46].…”

miR‐30b Promotes spinal cord sensory function recovery via the Sema3A/NRP‐1/PlexinA1/RhoA/ROCK Pathway

“…RhoA/ROCK pathway is recognized as a negative modulatory pathway that inhibits neuronal axon growth. Axon growth inhibitory factors, such as NogoA, exert their negative regulatory function via RhoA/ROCK pathway 11 . RhoA/ROCK pathway is reported to be downstream of sema3A 12 .…”

“…Axon growth inhibitory factors, such as NogoA, exert their negative regulatory function via RhoA/ROCK pathway. 11 RhoA/ROCK pathway is reported to be downstream of sema3A. 12 Therefore, inhibiting the Sema3A/NRP-1/PlexinA1/RhoA/ROCK pathway could be a novel treatment strategy to induce primary sensory neuronal axon growth post-SCI.…”