Cytoskeletal coordination during neuronal migration

Abstract

Discoveries from human and mouse genetics have identified cytoskeletal and signaling proteins that are essential for neuronal migration in the developing brain. To provide a meaningful context for these studies, we took an unbiased approach of correlative electron microscopy of neurons migrating through a three-dimensional matrix, and characterized the cytoskeletal events that occur as migrating neurons initiate saltatory forward movements of the cell nucleus. The formation of a cytoplasmic dilation in the proximal leading process precedes nuclear translocation. Cell nuclei translocate into these dilations in saltatory movements. Time-lapse imaging and pharmacological perturbation suggest that nucleokinesis requires stepwise or hierarchical interactions between microtubules, myosin II, and cell adhesion. We hypothesize that these interactions couple leading process extension to nuclear translocation during neuronal migration.

Fig. 1.

A cytoplasmic dilation within the leading process predicts the position into which the nucleus will move at the end of a saltatory movement. (A) Phase-contrast images of a neuron migrating in Matrigel (see Movie 1). As the leading edge advanced, a dilation (arrowhead) formed in the leading process (8:00 min) and was separated from the cell soma by a constriction. Between 8:00 and 14:00 min, the nucleus moved forward abruptly into the dilation, and the cycle started again with a new dilation apparent at 23:00. Times in all time-lapse images are represented in min:s. (B) A migrating neuron imaged at more frequent time intervals (see Movie 2). During this sequence, the neuron formed three discrete dilations (arrowheads, apparent in images captured at 0:00, 14:15, and 19:00) and performed two cell soma translocations in which the nucleus moved into the dilation. At 4:15, the leading edge paused (arrow), and subsequently (at 19:00) a dilation formed at this location (see also D). (C) Graph of the positions of the leading process (yellow), dilation (magenta), and cell soma (blue) of the neuron shown in A plotted against time. Note that, in most cases, the position of the dilation predicted the position into which the cell soma would move at a later time. (D) Graph depicting the movements of the neuron shown in B. In the sequence highlighted in pink, the leading process paused (4:00-5:00; see B). Later (approximately 18:00), a dilation formed at this position and subsequently (approximately 27:00) the nucleus moved into the same location (data not shown in B). (E) Phase-contrast image of a neuron with a prominent dilation that was fixed 30 seconds later (E′) as its nucleus initiated movement. This cell was then processed for transmission EM (panels F-I). (F) EM analysis of the cell in E′ revealed a multilobed nucleus (nuc) that seemed to be distorted by longitudinal arrays of microtubules. Individual microtubules seemed to be cross-linked by electron-dense bridges (Inset). (G) The microtubule arrays (arrowheads) emanated from the centrosome (arrow) and were aligned parallel to the direction of migration. (H) Long microtubules (arrowheads) occupied the regions in which the nucleus was deformed and indented. (I) Microtubules (arrowheads) were found in close proximity to the nuclear envelope (ne). The arrow marks a nuclear pore.

Fig. 2.

Nocodazole treatment induces forward nuclear movement and membrane blebbing. (A) A neuron that had formed a dilation but had not initiated nuclear movement was induced to move by the application of 1 μM nocodazole at 3:00 (see Movie 3). Note that the cell's leading edge remained largely stationary as the nucleus advanced forward. Times are represented in min:s. (B) A second example of a neuron that had formed a dilation before the addition of nocodazole, which induced nuclear movement (see Movie 4). This neuron's leading process continued to advance along the processes of other cells after drug addition, and the nucleus moved forward in a relatively continuous manner. Note the widespread membrane blebbing and contractions across the cell surface at and after 13:00. (C) The distances over which the leading process (yellow) and cell soma (blue) moved over time are plotted for the neuron and are shown in B. Red arrow indicates the time at which nocodazole was added. A characteristic lag of ≈10 min preceded the initiation of forward nuclear movement. Note the stable position of the leading process. (D) Plot of the distances that the leading process (yellow) and cell soma (blue) moved over time for the neuron shown in B. In this case, the leading process continued to extend after nocodazole addition (red arrow), and the nucleus moved forward with few or no pauses while advancing.

Fig. 3.

Membrane blebbing at the cell rear occurs in normal migrating neurons during nucleokinesis. (A) Time-lapse phase images of a neuron during nuclear translocation. Nuclear movement was accompanied by prominent membrane blebbing at the rear of the cell (arrowhead) (see Movie 5). (B) Correlative EM of a neuron undergoing nuclear movement. The nucleus of this cell had just begun to enter the dilation in the leading process. Higher-magnification views of this neuron are shown in C-F.(C-E) Membrane protrusions at the rear of the cell shown in B correlated with blebbing activity (C). By using mitochondria (asterisks) for orientation, adjacent sections revealed that blebbing occurred at or near regions in which the plasma membrane seemed to form contacts with the matrix (D). In some cases, densities within the membrane protrusions may correspond to adhesion puncta being broken away from the matrix (arrowheads in E and F). (G) This neuron showed a morphology characteristic of the late stages of nuclear movement, including a “bulb” of cytoplasm at its rear, similar to that seen in live cells (compare Fig. 4). The bulb was separated from the bulk of the cell soma by a constriction (arrowheads), which seemed to pinch the nucleus. (H and I) Higher magnification of adjacent sections of the neuron shown in G. The bulbous region at the cell rear was rich in microfilaments (arrowheads) but almost completely devoid of microtubules. (J) In contrast, the rear of a stationary neuron displayed long microtubules (arrowheads) present in the cell rear near matrix attachments.

Fig. 4.

Localization of activated non-muscle myosin II in normal and blebbistatin-treated neurons and effects of blebbistatin on motility. (A and B) Neurons were stained with antibodies to non-muscle myosin IIB (red) and serine-19 phosphorylated regulatory light chain (RLC) (green) and DAPI to reveal nuclei (blue in A). Activated myosin IIB was highly concentrated at the rear of cells that showed the characteristic morphology of migrating neurons, including one with a pinched or constricted nucleus in the process of translocating into a dilation (arrowheads). Although activated myosin IIB was present at the tips of most processes, it was not visible within the soma of nonmigratory neurons. For example, neurons that extended two processes (asterisk in B) were not observed to migrate during imaging sessions, and the somata of such cells did not display phospho-RLC labeling. (C and D) Neurons stained with antibodies to myosin IIB (red) and phospho-RLC (green) revealed the aggregation of activated myosin II (puncta) in the cell soma after exposure to blebbistatin. (E-G) Time-lapse images from a culture exposed to 50 μM blebbistatin (see Movie 6). The culture exhibited robust migration before drug addition at 48:00. After the addition of blebbistatin, the cell bodies of migrating neurons immediately stopped (arrows), although their leading processes (arrowheads) continued to advance exuberantly. Times are represented in min:s.

Fig. 5.

Blebbistatin rapidly inhibited nocodazole-induced membrane protrusions and soma translocation. (A) A neuron that displayed a prominent dilation was treated with 1 μM nocodazole at 9:15, which first induced nuclear movement and then vigorous global membrane blebbing. The addition of 100 μM blebbistatin at 39:45 rapidly inhibited motility (see Movie 7). Times are represented in min:s. (B) Simultaneous addition of 1 μM nocodazole and 100 μM blebbistatin at 9:15 to a neuron that displayed a cytoplasmic dilation inhibited both the nuclear movements and membrane blebbing that were induced by the addition of nocodazole alone (see Movie 8).

Fig. 6.

A model for cytoskeletal coordination during cycles of saltatory neuronal migration. As the leading process extends, it makes adhesive contacts with the extracellular matrix (blue). After the growth of the leading process past this position, a cytoplasmic dilation forms distal to the cell nucleus. Before the onset of nuclear movement, the centrosome (red dot) is moved by an as-yet unknown mechanism into the forming dilation. Microtubules (red lines) within the cell soma form longitudinal arrays (possibly as a result of pulling forces generated by centrosome movement) and vacate the cell rear. The nucleus then translocates toward the centrosome along the longitudinal microtubule arrays. We postulate that an absence of microtubules at the cell rear triggers contractions mediated by myosin II (yellow), which generates a pushing force on the nucleus and serves to break adhesions at the cell rear. Nuclear movement stops as the nucleus enters the former location of the dilation, and the process begins again.