Abstract
Migration and pathfinding of neuronal growth cones during neurite extension is critically dependent on dynamic microtubules. In this study we sought to determine, which aspects of microtubule polymerization relate to growth cone morphology and migratory characteristics. We conducted a multiscale quantitative microscopy analysis using automated tracking of microtubule plus ends in migrating growth cones of cultured murine dorsal root ganglion (DRG) neurons. Notably, this comprehensive analysis failed to identify any changes in microtubule polymerization parameters that were specifically associated with spontaneous extension vs. retraction of growth cones. This suggests that microtubule dynamicity is a basic mechanism that does not determine the polarity of growth cone response but can be exploited to accommodate diverse growth cone behaviors. At the same time, we found a correlation between growth cone size and basic parameters of microtubule polymerization including the density of growing microtubule plus ends and rate and duration of microtubule growth. A similar correlation was observed in growth cones of neurons lacking the microtubule-associated protein MAP1B. However, MAP1B-null growth cones, which are deficient in growth cone migration and steering, displayed an overall reduction in microtubule dynamicity. Our results highlight the importance of taking growth cone size into account when evaluating the influence on growth cone microtubule dynamics of different substrata, guidance factors or genetic manipulations which all can change growth cone morphology and size. The type of large scale multiparametric analysis performed here can help to separate direct effects that these perturbations might have on microtubule dynamics from indirect effects resulting from perturbation-induced changes in growth cone size.
Highlights
Continuous remodeling of the microtubule cytoskeleton has long been recognized as essential for neurite outgrowth and growth cone guidance in response to diverse attractive and chemo-repulsive guidance cues (Letourneau and Ressler, 1984; Challacombe et al, 1997; Kalil and Dent, 2005; Lowery and Van Vactor, 2009; Dent et al, 2011)
Quantitative properties of 783 migrating growth cones and microtubule polymerization properties of their associated 18169 YFP-EB3 comets were extracted using imaging at two different time scales (Figure 1, Figure S3 and Tables S1, S2)
In small and large MAP1B-null growth cones the steady decrease in comet speed with growth cone size mirrors what we found in wild-type growth cones
Summary
Continuous remodeling of the microtubule cytoskeleton has long been recognized as essential for neurite outgrowth and growth cone guidance in response to diverse attractive and chemo-repulsive guidance cues (Letourneau and Ressler, 1984; Challacombe et al, 1997; Kalil and Dent, 2005; Lowery and Van Vactor, 2009; Dent et al, 2011). With the discovery of proteins that bind to plus ends of polymerizing microtubules such as CLIP-170 (Perez et al, 1999) and end-binding (EB) protein 1 (Mimori-Kiyosue et al, 2000) it became more feasible to monitor microtubule dynamics in cultured mammalian neurons (Stepanova et al, 2003). These experiments were conducted on primary neurons transiently transfected or infected with constructs or viruses encoding fluorescent protein-tagged end-binding proteins. In combination with a recently developed microtubule plus-end tracking software (Matov et al, 2010; Applegate et al, 2011), large scale quantitative microscopy investigations of a potential relationship between microtubule dynamics and growth cone behavior are possible
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
