The predominant form in which neurofilament subunits undergo axonal transport varies during axonal initiation, elongation, and maturation

Neurofilament Polymer Transport in Axons

“…In neuronal cell lines, punctate structures predominate at early times after neurite initiation, whereas both punctate and filamentous structures are observed at later times (Yabe et al, 2001;Helfand et al, 2003). Based on these observations, Yabe et al (1999Yabe et al ( , 2001) have proposed that neurofilament proteins may be capable of movement in both polymerized and unpolymerized forms, that the predominant form varies depending on the differentiation state of the Figure 3. Electron microscopy of a captured filament using ultrathin sectioning.…”

Neurofilament Transport Is Dependent on Actin and Myosin

“…Although NFs undergo predominantly anterograde axonal transport (for review, see Shea and Yabe, 2000), many studies in culture and in situ also demonstrate that some NFs undergo retrograde subunit transport (Glass and Griffin, 1991;Watson et al, 1993;Koehnle and Brown, 1999;Yabe et al, 1999Yabe et al, , 2001aRoy et al, 2000;Shah et al, 2000;Wang et al, 2000). Retrograde NF transport may be mediated by the retrograde motor dynein (Shah et al, 2000).…”

“…In some cases we also used a construct encoding eGFP conjugated to the N terminal (generous gift from Dr. R. Liem, Columbia University, New York, NY), which, as described previously (Yabe et al, 2001a), yielded similar results; all images presented herein are derived from transfections with the use of C-terminally linked GFP.…”

“…We also monitored translocation of individual GFP-tagged particles by using two methods. Unlike microinjection in which the localization of bNF-L subunits can be visualized within minutes after injection, transfection under our conditions requires 18 -24 hr before sufficient GFP-tagged subunits have been expressed to allow reproducible visualization (Yabe et al, 1999(Yabe et al, , 2001a. Although GFP-tagged subunits typically have incorporated throughout the axonal NF network of many cells by this time, in those cells in which this has not yet occurred multiple GFP-tagged punctate and filamentous structures can be visualized readily along the periphery of axonal neurites.…”

“…The percentage of particles displaying either red or green fluorescence versus those displaying orange-yellow fluorescence was quantified at intervals before and after treatment. Translocation of GFP-tagged structures also was quantified by superimposing an arbitrary vertical line across the identical location in sequential images, which allowed us to determine the number of particles that had undergone either anterograde or retrograde translocation or had displayed no net translocation during a given interval; particles were considered to have undergone translocation provided they had shifted at least one full particle diameter between intervals (Yabe et al, 2001a).…”