Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones

Abstract

We have used multimode fluorescent speckle microscopy (FSM) and correlative differential interference contrast imaging to investigate the actin–microtubule (MT) interactions and polymer dynamics known to play a fundamental role in growth cone guidance. We report that MTs explore the peripheral domain (P-domain), exhibiting classical properties of dynamic instability. MT extension occurs preferentially along filopodia, which function as MT polymerization guides. Filopodial bundles undergo retrograde flow and also transport MTs. Thus, distal MT position is determined by the rate of plus-end MT assembly minus the rate of retrograde F-actin flow. Short MT displacements independent of flow are sometimes observed. MTs loop, buckle, and break as they are transported into the T-zone by retrograde flow. MT breakage results in exposure of new plus ends which can regrow, and minus ends which rapidly undergo catastrophes, resulting in efficient MT turnover. We also report a previously undetected presence of F-actin arc structures, which exhibit persistent retrograde movement across the T-zone into the central domain (C-domain) at ∼1/4 the rate of P-domain flow. Actin arcs interact with MTs and transport them into the C-domain. Interestingly, although the MTs associated with arcs are less dynamic than P-domain MTs, they elongate efficiently as a result of markedly lower catastrophe frequencies.

Actin Aggregations Mark the Sites of Neurite Initiation

“…It has been proposed that microtubule-associated proteins that can also interact with actin, such as MAP2c, promote neurite extension by facilitating microtubule assembly and extension at the sites of actin aggregates [34,38,39,41,47,78,79]. Similar effects have also been reported for the F-actin-associated protein drebrin, which has recently been shown to bind to the microtubule plusend protein EB3 and also promote neuritogenesis [80].…”

HDAC6 and RhoA are novel players in Abeta-driven disruption of neuronal polarity

“…Therefore, an alteration in developmental processes that are fundamental for the correct migration of neurons might be at the basis of cell death in the areas of adult neurogenesis. These processes depend on coordinated action of microfilaments and MT 40 , prompting further investigation on the effect of Ab on actin-MT crosstalk.…”

“…Moreover, the heterodimeric fraction was further increased due to MT instability and to the inhibitory effect of Ab on HDAC6. Increased myosin II-driven contractility together with MT instability in the axon would further exacerbate the tendency of the growth cone to collapse 40 , phenomenon that would affect growth cones motility in developing neurons or synapses stability in neuronal networks. Our data on the accumulation of ac-tub heterodimers might explain controversial data in previous reports on the level of tubulin acetylation.…”

Permeation of Fluorophore-Conjugated Phalloidin into Live Hair Cells of the Inner Ear Is Modulated by P2Y Receptors

“…Upon permeating hair cells, it specifically labels F-actin-rich cellular structures, including stereocilia, and cuticular plates. Due to phalloidin's affinity for F-actin, it is useful for investigating retrograde F-actin flow and the coordination between the actin and microtubules cytoskeletal networks advancing growth cones and neurites (Schaefer et al 2002;Burnette et al 2007Burnette et al , 2008Schaefer et al 2008). If a low enough concentration of fluorophore-conjugated phalloidin is introduced into HCs, this could provide a new method for analyzing retrograde actin flow in HCs and treadmilling rates within stereocilia (Schneider et al 2002;Zhang et al 2012).…”

“…This provides dynamic measures of localized directions and rates of Factin flow (Lin and Forscher 1995;Waterman-Storer et al 1998;Schaefer et al 2002Schaefer et al , 2008Burnette et al 2008). …”