The Hatten Lab Projects

Cytoskeletal Organization of the Migrating Neuron



Previous studies of microtubules in migrating neurons revealed a cage-like distribution of microtubules around the nucleus and a longitudinally-oriented system of microtubules extending to the tip of the leading process. By contrast, actin filaments, visualized by rhodamine-phalloidin labeling, formed a subcortical rim underneath the plasma membrane of the cell soma, with actin filaments heavily concentrated in the lamellipodia of the leading process. Disruption of either the microtubule system, by treatment with nocodozole, or the actin system, by treatment with cytochalasin, halted neuronal migration. These results underscore the importance of cytoskeletal dynamics to glial-guided migration (Rivas and Hatten, 1996).
Microtubule organization in migrating granule cells.
Granule neurons migrating on laminin-coated glass fibers were fixed, stained by indirect immunofluorescence with a monoclonal antibody to b-tubulin, and examined by confocal microscopy . a, Nomarski micrograph of migrating granule neuron. The direction of migration is towards the top of the panel. Optical sections started 1.5µm from the top of the cell body and were taken towards the glass coverslip; optical sections are 1.0µm apart. note the cage-like network of microtubules in the cell body. Spiraling microtubules are be seen coursing down the leading process (double arrow in c and d), Scale bar, 4µm.
Presently, we are focused on obtaining a deeper understanding of the dynamics of neuronal migration by imaging the cytoskeletal dynamics in migrating neurons. New fixation methods illustrate the "cage-like" tubulin structure surrounding the nucleus (left, and see insert at upper right), as well as a network of microtubules within the leading process of the migrating neuron.

Recently, we have used retroviral constructs containing the reporter Venus, a mutated form of YFP that is 10-20 times brighter than EGFP, to express fluorescently tagged -tubulin, p50- dynactin, and mPAR6a in migrating neurons. We have shown that the stability of the tubulin in the "cage" is similar in migrating and stationary neurons, and discovered that mPAR6a marks the centrosome in the migrating neurons. By real time imaging methods, we have been able to demonstrate that the centrosome moves forward just before the nucleus advances, providing a two-stroke engine that propels the neuron on the glial guide. This marks a major advance in our understanding of the mechanism of neuronal migration, suggesting that pathways that control the cytoskeleton modulate neuronal migration, with adhesion systems playing a permissive rather than instructive role in this vital process (Solecki, et al, submitted).
mPar6 alpha and p50 dynactin label the neuronal centrosome. (A) The localization Venus-mPar6 and p50 dynactin within live cerebellar granule neurons. In live neurons, Venus-mPar6 and p50 dynactin both strongly label a single punctate organelle just forward of the nucleus. Each image is a maximum projection of a z-stack. (B) Venus-mPar6 is concentrated near the centrosome. Cerebellar granule neurons expressing Venus-mPar6 were immunostained with anti-GFP and anti-gamma tubulin antibodies. An mPar6 labeled structure co-localizes with gamma tubulin immuno-reactivity is the centrosome. (C) Endogenous mPar6 immuno-reactivity also co-localizes with ?-tubulin indicating that the localization of Venus-mPar6? mimics that of the endogenous protein. (D) Purified granule neurons were immunostained with anti-PKC zeta and anti gamma tubulin antibodies. PKC zeta immunoreactivity is concentrated near gamma tubulin a marker of the centrosome. (E) Cerebellar granule neurons were immuno-stained with anit-p50 dynactin and anti alpha tubulin antibodies. The predominant p50 dynactin labeled structure appears at points where the microtubule cytoskeleton is nucleated suggesting that the structure labeled in Panel A is the centrosome. Significant nuclear immunoreactivity was also observed. Arrowheads indicate the spindle poles of a mitotic granule neuron precursor and full arrows indicate the centrosomal p50 Dynactin. Scale bars equal 10 micron. The mPar6 and p50 dynactin images share the same scale.
David Solecki, Nature Cover During development, neurons migrate along glial fibers to take their place within the cortical layers. Hatten and colleagues show that overexpression of mPar6 alpha disrupts the coordinated cytoskeletal mechanisms that enable this migration. The cover image shows young cerebellar granule cells migrating along Bergmann glia fibers. The centrosome and nuclei are labeled with dynein intermediate chain (red), and the perinuclear microtubule cage is marked with beta-tubulin (green). In the migrating neurons, the centrosome is positioned forward of the nucleus.
Image rendered by Nick Didkovsky.
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