Computer vision profiling of neurite outgrowth dynamics reveals spatiotemporal modularity of Rho GTPase signaling

Fusco, Ludovico; Lefort, Riwal; Smith, Kevin; Benmansour, Fethallah; González, Germán; Barillari, Caterina; Rinn, Bernd; Fleuret, François; Fua, Pascal; Pertz, Olivier

doi:10.1083/jcb.201506018

Cited by 21 publications

(33 citation statements)

References 46 publications

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“…However, this simplified view is not consistent with the finding that multiple GEFs, GAPs, and effectors outnumber their cognate Rho GTPases [58][59][60] , suggesting a more complex scenario in which multiple spatiotemporal Rho GTPase signaling networks (including GAP, GEFs, GTPases, and effectors) modulate different morphological processes. A recent observation that different RhoA-specific GAPs regulate two distinct RhoA signaling complexes lends support to this hypothesis: while ARHGAP5 is involved in the RhoA-mediated growth cone collapse, RhoA/ DLC1 might trigger mDia1 formin to polymerize F-actin and to enable filopodia extension 61 We have recently reported that a distinct serotonin receptor type, 5-HT 7 R, prompts morphological effects opposite to those reported here for the 5-HT 4 R: activation of the 5-HT 7 R facilitates Hippocampal neurons (DIV12) isolated from WT (a) or 5-HT 4 R-deficient mice (KO) (b) were treated with the 5-HT 4 R agonist BIMU8 and/or antagonist GR with or without pre-treatment with the specific ROCK inhibitor Y-27632 (50 µM), followed by the western blot with antibodies against phosphorylated (upper row, p-Cof), total cofilin (middle, Cof) and GAPDH as a loading control (bottom). (Left) Representative western blot showing cofilin phosphorylation in WT neurons (a) and in 5-HT 4 R KO neurons (b).…”

Section: Discussionmentioning

confidence: 93%

Serotonin 5-HT4 receptor boosts functional maturation of dendritic spines via RhoA-dependent control of F-actin

et al. 2020

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Activity-dependent remodeling of excitatory connections underpins memory formation in the brain. Serotonin receptors are known to contribute to such remodeling, yet the underlying molecular machinery remains poorly understood. Here, we employ high-resolution time-lapse FRET imaging in neuroblastoma cells and neuronal dendrites to establish that activation of serotonin receptor 5-HT 4 (5-HT 4 R) rapidly triggers spatially-restricted RhoA activity and G13-mediated phosphorylation of cofilin, thus locally boosting the filamentous actin fraction. In neuroblastoma cells, this leads to cell rounding and neurite retraction. In hippocampal neurons in situ, 5-HT 4 R-mediated RhoA activation triggers maturation of dendritic spines. This is paralleled by RhoA-dependent, transient alterations in cell excitability, as reflected by increased spontaneous synaptic activity, apparent shunting of evoked synaptic responses, and enhanced long-term potentiation of excitatory transmission. The 5-HT 4 R/G13/RhoA signaling thus emerges as a previously unrecognized molecular pathway underpinning usedependent functional remodeling of excitatory synaptic connections.

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Section: Discussionmentioning

confidence: 93%

Serotonin 5-HT4 receptor boosts functional maturation of dendritic spines via RhoA-dependent control of F-actin

et al. 2020

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“…Another example is the regulation of the exquisitely focused RhoA activity pattern at the tip of F-actin bundles that form neuronal growth cone filopodia ( Figure 1D ). A recent study identified the RhoA-specific GAP DLC1 (deleted in liver cancer 1) to spatially regulate the filopodial RhoA activity pattern 37 . RNA interference (RNAi)-mediated DLC1 knockdown leads to widening of the RhoA activity domain, suggesting that DLC1 acts by shaping the focused RhoA activity zone at filopodial tips.…”

Section: Gef/gap-mediated Rho Gtpase Fluxes Underlie Spatio-temporal mentioning

confidence: 99%

“…Almost all GEFs bear a lipid-interaction domain 37 , 39 , 40 : a pleckstrin homology (PH), a DOCK homology region 1 (DHR-1), or a Bin-Amphiphysin-Rvs (BAR) domain 41 – 43 . Many GAPs also contain a variety of lipid-binding domains 44 .…”

Section: Spatio-temporal Regulation Of Gefs and Gapsmentioning

confidence: 99%

The dynamics of spatio-temporal Rho GTPase signaling: formation of signaling patterns

Fritz

Pertz

2016

F1000Res

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Rho GTPases are crucial signaling molecules that regulate a plethora of biological functions. Traditional biochemical, cell biological, and genetic approaches have founded the basis of Rho GTPase biology. The development of biosensors then allowed measuring Rho GTPase activity with unprecedented spatio-temporal resolution. This revealed that Rho GTPase activity fluctuates on time and length scales of tens of seconds and micrometers, respectively. In this review, we describe Rho GTPase activity patterns observed in different cell systems. We then discuss the growing body of evidence that upstream regulators such as guanine nucleotide exchange factors and GTPase-activating proteins shape these patterns by precisely controlling the spatio-temporal flux of Rho GTPase activity. Finally, we comment on additional mechanisms that might feed into the regulation of these signaling patterns and on novel technologies required to dissect this spatio-temporal complexity.

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“…Fluorescent biosensors are ideal tools to examine the activity of molecules at the subcellular level, in the context of real-time cellular behaviors. When combined with computational image analysis and statistical modeling, biosensors can provide quantitative insights into how molecules interact in space and time to generate specific biological behaviors ( Machacek et al, 2009 ; Fusco et al, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

Spatiotemporal dynamics of GEF-H1 activation controlled by microtubule- and Src-mediated pathways

Azoitei

Noh

Marston

et al. 2019

Journal of Cell Biology

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Rho family GTPases are activated with precise spatiotemporal control by guanine nucleotide exchange factors (GEFs). Guanine exchange factor H1 (GEF-H1), a RhoA activator, is thought to act as an integrator of microtubule (MT) and actin dynamics in diverse cell functions. Here we identify a GEF-H1 autoinhibitory sequence and exploit it to produce an activation biosensor to quantitatively probe the relationship between GEF-H1 conformational change, RhoA activity, and edge motion in migrating cells with micrometer- and second-scale resolution. Simultaneous imaging of MT dynamics and GEF-H1 activity revealed that autoinhibited GEF-H1 is localized to MTs, while MT depolymerization subadjacent to the cell cortex promotes GEF-H1 activation in an ~5-µm-wide peripheral band. GEF-H1 is further regulated by Src phosphorylation, activating GEF-H1 in a narrower band ~0–2 µm from the cell edge, in coordination with cell protrusions. This indicates a synergistic intersection between MT dynamics and Src signaling in RhoA activation through GEF-H1.

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Computer vision profiling of neurite outgrowth dynamics reveals spatiotemporal modularity of Rho GTPase signaling

Abstract: NeuriteTracker is a computer vision approach used to analyze neuronal morphodynamics and to examine spatiotemporal Rho GTPase signaling networks regulating neurite outgrowth.

Cited by 21 publications

References 46 publications

Serotonin 5-HT4 receptor boosts functional maturation of dendritic spines via RhoA-dependent control of F-actin

Serotonin 5-HT4 receptor boosts functional maturation of dendritic spines via RhoA-dependent control of F-actin

The dynamics of spatio-temporal Rho GTPase signaling: formation of signaling patterns

Spatiotemporal dynamics of GEF-H1 activation controlled by microtubule- and Src-mediated pathways

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