1. Long-Range Ca2+ Signaling from Growth Cone to Soma Mediates Reversal of Neuronal Migration Induced by Slit-2 
Chen-bing Guan, Hua-tai Xu, Ming Jin, Xiao-bing Yuan, Mu-ming Poo 
Cell 
Volume 129, Issue 2, Pages (April 2007)
DOI: /j.cell
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1
Dominant Role of the Growth Cone in Sensing the Slit-2 Gradient
(A) Images of a migrating cerebellar granule cell at various times (number in min) before and after frontal application of a Slit-2 gradient are shown.
(B) shows changes in the rate of growth-cone extension and soma translocation after frontal application of the Slit-2 gradient (n = 10). Dotted lines mark the onset of detectable changes. Data represent mean ± SEM.
(C) Images show a neuron after application of the Slit-2 gradient in front of the soma.
(D) The average rate (± SEM, n = 10) of the extension of leading and trailing neurites and of soma movement under various conditions is shown.
(E) Example of a cultured granule neuron cotransfected with dsRed and GFP-tagged Robo2 is shown. The fluorescence intensity ratio of Robo2-GFP to dsRed represents the Robo2 density normalized by the thickness of the cell and coded by pseudocolors in a linear scale (bar on the right). Scale is 10 μm.
Cell  , DOI: ( /j.cell )
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3. Figure 2
Frontal Application of the Slit-2 Gradient Induces a Ca2+ Wave from Growth Cone to Soma
(A) Images show the neuronal [Ca2+]i at various times (0 to 60 s) after frontal application of a Slit-2 gradient (arrowheads). The [Ca2+]i was determined by the ratio of Fluo-4 to Fura-Red fluorescence and coded by pseudocolors in a linear scale.
(B) Fractional changes in [Ca2+]i over time before and after 1 min exposure to a frontal Slit-2 gradient are shown. Data represent mean ± SEM (n = 11).
(C and D) Measurements of [Ca2+]i similar to those in (A) and (B) are shown, except that the Slit-2 gradient was applied for 1 min to the trailing neurite (arrow heads). Data represent mean ± SEM.
(E and F) Similar experiment as in (A) and (B) is shown, with [Ca2+]i monitored at a higher time resolution, revealing a delay in the [Ca2+]i rise at the soma (dotted lines in F, n = 18). Scale is 10 μm. Data represent mean ± SEM.
Cell  , DOI: ( /j.cell )
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4. Figure 3 Ca2+ Wave Mediates the Reversal of Soma Translocation
(A) Images show [Ca2+]i in a neuron before and after the frontal Slit-2 application in the presence of ryanodine at a high concentration (0.5 mM) in the bath.
(B) Ca2+ changes at the leading growth cone and the soma are shown 1 min after frontal Slit-2 application in cultures treated with (n = 12) or without (n = 11) the high-level ryanodine. Data represent mean ± SEM (asterisk, p < 0.05, Student's t test).
(C) Images of a neuron show growth-cone collapse without the reversal of soma translocation, following the application of frontal gradient Slit-2 in the presence of local perfusion of a high-level ryanodine (2 mM) at the soma (white pipettes).
(D) Final mean soma migration rate under frontal gradient of low-level ryanodine (10 μM in the pipette, n = 19) or soma perfusion with high-level ryanodine with (n = 12) or without (n = 10) frontal Slit-2 gradient is shown. Data represent mean ± SEM.
(E) Frontal gradient of low-level ryanodine resulted in the reversal of soma movement without inducing the immediate collapse of the leading growth cone. Scale is 10 μm.
(F) Ca2+ elevation in the leading growth cone and the soma induced by the frontal gradient of low-level ryanodine (n = 16) is shown. Data represent mean ± SEM.
Cell  , DOI: ( /j.cell )
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5. Figure 4
Requirement of RhoA Activity in Ca2+-Mediated Migration Reversal
(A) Example shows results of pull-down assays confirming the activity of mutated RhoGTPase proteins in HEK293 cells. The GTP-bound RhoGTPases (active form) were shown by blotting protein precipitates with glutathione-Sepharose beads, which were prebound with GST-fused RhoGTPase-binding domain. (Left) Western blots showing cells transfected with DN-RhoA and CA-RhoA exhibited a lower and higher level of active RhoA (asterisk), respectively, than that of WT-RhoA with equal input of total RhoA (lower lane). (Right) The same assay is shown for cells transfected with Cdc42 constructs. Similar results were obtained in at least three independent experiments.
(B) Example shows a neuron expressing DN-RhoA-GFP. Frontal application of Slit-2 gradient led to the collapse of the growth cone and the inhibition of soma translocation without reversal. Insert shows fluorescence image of the same cell.
(C) Example shows a neuron expressing DN-Cdc42-GFP, showing Slit-2-induced reversal of migration, with the fluorescent image of the cell in the insert. Scale is 10 μm.
(D) Summary of the final rate of soma translocation after frontal application of a gradient of Slit-2 or low-level ryanodine (10 μM in pipette) is shown under various conditions. Toxin B (10 ng/ml, n = 16), C3 transferase (5 μg/ml, n = 17), or Y27632 (10 μM, n = 16 for Slit-2; n = 11 for low-level ryanodine) was added in the bath. Data represent mean ± SEM (n = 17 for control; n = 10 for DN-RhoGTPases).
(E) Example results of pull-down assays of RhoA activity in cultured granule cells under various manipulations of [Ca2+]i are shown. The western blots depict downregulation of active RhoA (asterisk) by Slit-2 treatment for 5 min (lower panel) or by direct [Ca2+]i elevation by low-level ryanodine (10 nM) for 3 min (upper panel). Downregulation was absent in cells preincubated with BAPTA-AM (10 μM). The lower blots of two panels show total RhoA.
(F) Slit-2-induced downregulation of RhoA activity (indicated by the ratio of active RhoA to total RhoA) is shown. Data represent mean ± SEM (n = 8, asterisk, p < 0.05, ANOVA).
Cell  , DOI: ( /j.cell )
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6. Figure 5 Polarized Distribution of RhoA in Migrating Neurons
(A) Images show migrating neurons expressing the GFP-tagged WT RhoA, Rac1, or Cdc42 (time in min).
(B) Distribution of the three GFP-RhoGTPases in the soma is shown. The fluorescence intensity was measured along the somatic axis (front to rear) over 20 equal segments. The value obtained at each segment (F) was normalized as the fractional difference given by (F-F0)/F0, where F0 is the average intensity for all 20 segments (n = 14). Data represent mean ± SEM.
(C) A migrating neuron expressing WT-RhoA-GFP exhibited RhoA redistribution during the reversal of soma translocation triggered by the Slit-2 gradient.
(D) Distribution of WT-RhoA-GFP across the soma of the cell shown in (C) is shown at various times before and after the Slit-2 application.
(E) The reversal of soma translocation and redistribution of WT-RhoA-GFP induced by Slit-2 were prevented by the presence of high-level ryanodine (0.5 mM) in the bath. Scale is 10 μm.
(F) Summary shows all results on the RhoA distribution in the soma before and after frontal application of the Slit-2 or low-level ryanodine. Also shown are results for frontal Slit-2 gradient in the presence of high-level ryanodine in the bath. Asymmetry Index is defined as the difference between the average fluorescence intensities at the front (F1) and rear (F4) quarter of the soma divided by the sum of the fluorescence intensities at these two regions [(F1-F4)/(F1+F4)]. Data represent mean ± SEM (n = 8).
Cell  , DOI: ( /j.cell )
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7. Figure 6 Distribution of Active RhoA in Migrating Granule Cells
(A) Changes in the FRET signal in cultured granule cells during the first 15 min after the application of LPA (10 μg/ml, n = 12) or PBS (n = 30) are shown, confirming the effectiveness of the FRET probe in detecting RhoA activity. Data represent mean ± SEM.
(B) A migrating granule cell displays asymmetric active RhoA across the cell. FRET signals were coded by pseudocolors (bar on the right).
(C) Redistribution of active RhoA during the reversal of soma translocation was induced by a continuous frontal application of a Slit-2 gradient (time in minutes). Scale is 10 μm.
(D) Distribution of active RhoA in the soma before and after frontal application of Slit-2 or of low-level ryanodine (n = 8 each) is shown. Asymmetry Index is defined in Figure 5F. Data represent mean ± SEM.
Cell  , DOI: ( /j.cell )
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8. Figure 7
A Model for Ca2+/RhoA-Mediated Reversal of Neuronal Migration Induced by Slit-2
A migrating granule cell exhibits a front-to-rear gradient in the distribution of RhoA (Left). Frontal exposure to Slit-2 gradient leads to Ca2+ elevation in the growth cone and subsequent propagation of a Ca2+ wave to the soma, leading to a redistribution of RhoA (Middle), whose activity is required for the reversal of neuronal migration (Right).
Cell  , DOI: ( /j.cell )
Copyright © 2007 Elsevier Inc. Terms and Conditions