doi: 10.3389/fncel.2014.00400. eCollection 2014.
Capture of microtubule plus-ends at the actin cortex promotes axophilic neuronal migration by enhancing microtubule tension in the leading process
Affiliations
- PMID:25505874
- PMCID: PMC4245908
- DOI: 10.3389/fncel.2014.00400
Item in Clipboard
Capture of microtubule plus-ends at the actin cortex promotes axophilic neuronal migration by enhancing microtubule tension in the leading process
B Ian Hutchins et al. Front Cell Neurosci..
Display options
Format
Display options
Format
Abstract
Microtubules are a critical part of neuronal polarity and leading process extension, thus microtubule movement plays an important role in neuronal migration. However, the dynamics of microtubules during the forward movement of the nucleus into the leading process (nucleokinesis) is unclear and may be dependent on the cell type and mode of migration used. In particular, little is known about cytoskeletal changes during axophilic migration, commonly used in anteroposterior neuronal migration. We recently showed that leading process actin flow in migrating GnRH neurons is controlled by a signaling cascade involving IP3 receptors, CaMKK, AMPK, and RhoA. In the present study, microtubule dynamics were examined in GnRH neurons. Failure of the migration of these cells leads to the neuroendocrine disorder Kallmann Syndrome. Microtubules translocated forward along the leading process shaft during migration, but reversed direction and moved toward the nucleus when migration stalled. Blocking calcium release through IP3 receptors halted migration and induced the same reversal of microtubule translocation, while blocking cortical actin flow prevented microtubules from translocating toward the distal leading process. Super-resolution imaging revealed that microtubule plus-end tips are captured at the actin cortex through calcium-dependent mechanisms. This work shows that cortical actin flow draws the microtubule network forward through calcium-dependent capture in order to promote nucleokinesis, revealing a novel mechanism engaged by migrating neurons to facilitate movement.
Keywords: EB1; IP3 receptors; actin cytoskeleton; microtubules; neuronal migration; neuronal migration disorders; super resolution microscopy.
Figures
Microtubule dynamics during neuronal migration.(A) Microtubules in a migrating GnRH neuron (raw fluorescence left, scale bar, 5 μm; mid, nucleus outlined with dashed line; solid line indicates region for generating kymograph; image of the same cell at the end of the imaging session, right).(B) The microtubules translocate forward during neuronal migration (kymograph, duration 10 min). (C, left) Lower magnification view of fluorescent microtubule staining in a migrating GnRH neuron. Inset, region of higher-magnification region shown in pseudocolored time-lapse images (right). Scale bar, 10 μm. (C, right) Simultaneous forward microtubule translocation (arrow) and convergence with the soma (asterisk). Dotted lines show the distance between the soma edge and microtubule bundles at the beginning (blue) and end (green) of the imaging session; brackets (right) summarize the change in these distances from beginning (pre, 3.9 μm) to end (post, 2.7 μm).(D–F) Frame-by-frame analysis was performed (n = 65) on 2-min frames from 12 neurons (N = 9 explants).(D) Forward translocation of microtubules vs. soma speed within 2-min time frames (p < 0.0001, linear regression,r = 0.68).(E) Microtubule/soma convergence vs. soma speed shows only a weak relationship (p = 0.032, linear regression,r = 0.27).(F) Microtubule/soma convergence accounts for much of the residual soma speed after subtracting the effect of microtubule translocation rates (p < 0.0001, linear regression,r = 0.68).
Models to explain convergence between nucleus and leading process microtubules.(A) Testable model 1 (“Brake”): Microtubules act as a brake. Nucleokinesis is due to pushing forces from behind that cause the nucleus to “crash” against leading process microtubules. Leading process microtubules (green) form a barrier that slows (resistive force shown as arrow) the front edge of the nucleus (blue) as these compress together. In this model microtubule convergence and excess speed of the front edge are inversely correlated, and this convergence only occurs during nucleokinesis.(B) Testable model 2 (“Cable”): Microtubule motor proteins (black dots) draw the nucleus forward along the leading process microtubules, which can be thought of as cables or rails, as in other cell types (Zhang et al., 2009). The pulling force (arrow) from in front of the nucleus draws the front edge along microtubules faster than the center, causing elongation of the nucleus. In this model, microtubule/soma convergence and excess speed of the front edge are directly correlated, and convergence may also occur in GnRH neurons that have stalled. (C, left) Fluorescent staining of microtubules in a GnRH neuron that is not migrating. (C, right) Outlines indicate the border of the cell (solid) and nucleus (dotted), while the line shows the region measured for kymographs. Scale bar, 5 μm.(D) Backward microtubule translocation (arrows) in a paused neuron. Dotted lines show the distance between the soma edge and microtubule bundles at the beginning (blue) and end (green) of the imaging session; dashed blue lines denote the nucleus; brackets (right) summarize the change in these distances from beginning (pre, 6.1 μm) to end (post, 2.7 μm). Scale bar, 5 μm.(E) Kymograph of the region shown in(C), with an asterisk and arrow corresponding to the marked regions in(D); the microtubule bundle in the leading process (arrow) and the front edge of the soma (asterisk).(F) Schematic: During stalling, microtubules (green) reverse direction and move toward the nucleus.(G) Measurements of microtubule/soma convergence vs. excess speed of the front edge show a direct relationship (p = 0.0325, linear regression,r = 0.27,n = 65 frames from 12 neurons,N = 9 explants), refuting the “brake” model in(A) and supporting the “cable” model in(B).(H) Acute nocodazole (at microtubule depolymerizing concentrations) reduced neuronal migration rates in DIC-imaged GnRH neurons by 22% (p = 0.0051, Wilcoxon matched pairs signed rank test;n = 107 neurons fromN = 4 explants).
Inhibiting IP3 receptors reverses forward translocation of microtubules.(A,B) Fluorescent staining of microtubules in a migrating GnRH neuron.(B) Same as(A), but with a dotted lineshowing the border of the nucleus and a solid line showing the region shown for kymographs.(C,D) Forward translocation of microtubules (C, arrows) is reversed after application of 2-APB to block IP3 receptors (D, arrows). Dotted lines show the distance between the soma edge and microtubule bundles at the beginning (blue) and end (green) of the imaging session; brackets (right) summarize the change in these distances from beginning (pre, 3.4 μm forC and 3.6 μm forD) to end (post, 3.6 μm forC and 2.3 μm forD). Images in(A–F) are from the same cell.(E,F) Kymographs of the region shown in(B), containing the microtubule bundle (arrow) and edge of the soma (asterisk) as shown in(C,D).(G) Change in microtubule translocation rate corresponds to the change in soma speed (n = 35 frames from 7 neurons inN = 5 explants,p < 0.0001,r = 0.65, linear regression). Scale bars, 5 μm.
Microtubule +TIPs lose association with cortical actin after blocking calcium release.(A) Triple-staining against EB1 (green), F-actin (red), and GnRH (blue) in a control-treated cell with EB1 and F-actin imaged with STED microscopy.(B) Zoomed image of the box in(A). Arrows indicate examples of super-resolution co-localization, showing several EB1 puncta associated with the actin cortex in the leading process.(C,D) Low(C) and high magnification (D, of region boxed inC) of triple-stained neurons treated with 2-APB. Blocking calcium release reduces the number of EB1 puncta associated with the actin cortex (arrows,D). Scale bars, 5 μm.(E) No differences were detected in raw EB1 staining fluorescence (n.s.,p = 0.227,t-test, Cohen'sd = 0.53,n = 10 control and 12 2-APB treated GnRH neurons).(F) Fewer EB1 puncta were observed in the leading process actin cortex of 2-APB treated GnRH neurons compared with vehicle controls (***p = 0.0006,t-test, Cohen'sd = 1.75,n = 10 controls and 12 treated GnRH neurons,N = 3 explants for both conditions).(G) Fraction of time microtubules spent moving toward the distal growth cone was reduced by the inhibitor of cortical actin flow, Concanavalin A (n = 7 cells fromN = 3 explants;**p = 0.0026, Fisher's exact test).
References
- Canman J. C., Bement W. M. (1997). Microtubules suppress actomyosin-based cortical flow in Xenopus oocytes. J. Cell Sci. 110(Pt 1), 1907–1917. - PubMed
Grants and funding
LinkOut - more resources
Full Text Sources
Other Literature Sources