Microtubules and motor proteins support zebrafish neuronal migration by directing cargo

Abstract

Neuronal migration during development is necessary to form an ordered and functional brain. Postmitotic neurons require microtubules and dynein to move, but the mechanisms by which they contribute to migration are not fully characterized. Using tegmental hindbrain nuclei neurons in zebrafish embryos together with subcellular imaging, optogenetics, and photopharmacology, we show that, in vivo, the centrosome’s position relative to the nucleus is not linked to greatest motility in this cell type. Nevertheless, microtubules, dynein, and kinesin-1 are essential for migration, and we find that interference with endosome formation or the Golgi apparatus impairs migration to a similar extent as disrupting microtubules. In addition, an imbalance in the traffic of the model cargo Cadherin-2 also reduces neuronal migration. These results lead us to propose that microtubules act as cargo carriers to control spatiotemporal protein distribution, which in turn controls motility. This adds crucial insights into the variety of ways that microtubules can support successful neuronal migration in vivo.

In vivo photocontrol of microtubule dynamics and integrity, migration and mitosis, by the potent GFP-imaging-compatible photoswitchable reagents SBTubA4P and SBTub2M

“…(For example, 561 nm RFP imaging on the confocal microscope photoisomerises azobenzene PST-1 despite its extinction coefficients being < 30 cm −1 M −1 . 21 ) However, the SBTubs ’ cutoff suggested they would indeed be GFP-orthogonal: which was later confirmed and found to be crucial for in vivo use (see below).…”

“…Though the actin cytoskeleton is the major driver of cell migration, MTs are integral to directional migration and leading edge stabilisation 21,40,41 (as well as to proliferation): so we expected the single-cell motility as well as cell division rates could be locally affected. Therefore, we wished to test if repeated localized photoactivations of SBTubA4P (every 7 min) could be used over long timescales (>24 h) to inhibit outgrowth of light-targeted organoid branches, while leaving other branches of the same organoids to develop: so shaping and modulating cell migration and invasion with spatiotemporal control.…”

“…In the MT field, recent photoswitchable analogues of taxane 12 , epothilone 13 and colchicinoid 5,[14][15][16][17][18][19] MT inhibitors have all been applied to cellular studies. These photopharmaceuticals have enabled noninvasive, reversible optical control over MT dynamics and MT-associated downstream effects, with cell-specific spatial precision and sub-second-scale temporal precision, and have been brought to bear on research in embryology 20 , neuroscience 21 , and cytoskeleton 5 .…”

In Vivo Photocontrol of Microtubule Dynamics and Integrity, Migration and Mitosis, by the Potent GFP-Imaging-Compatible Photoswitchable Reagents SBTubA4P and SBTub2M

“…Based on the good performance of SBTubA4P in 2D cell culture, we assessed whether SBTubA4P can be controlled similarly precisely in a 3D organoid model, aiming to manipulate organoid development by locally interfering with the invasion of individual branches during the elongation phase. 42 Though the actin cytoskeleton is the major driver of cell migration, MTs are integral to directional migration and leadingedge stabilization 24,44,45 (as well as to proliferation): so we expected the single-cell motility as well as cell division rates could be locally affected. Therefore, we wished to test if repeated localized photoactivations of SBTubA4P (every 7 min) could be used over long timescales (>24 h) to inhibit outgrowth of lighttargeted organoid branches, while leaving other branches of the same organoids to develop: so shaping and modulating cell Journal of the American Chemical Society pubs.acs.org/JACS Article Journal of the American Chemical Society pubs.acs.org/JACS Article migration and invasion with spatiotemporal control.…”

Photocontrolling Microtubule Dynamics with Photoswitchable Chemical Reagents

“…SBTubs have shown some ability to apply subcellularly-localised effects in large neurons [7]. PSTs have been used to reversibly block/unblock mitosis in worm embryo [2]; manipulate MT dynamics and organising structure in the mouse embryo [27,28], tissue mechanics in the developing fly [29], redirect migrating cells in culture [30], locally affect neurons [8], and block migrating neurons in vivo in zebrafish [31]. Note 14.…”