Antagonistic Forces Generated by Cytoplasmic Dynein and Myosin‐II during Growth Cone Turning and Axonal Retraction

Abstract

Cytoplasmic dynein transports short microtubules down the axon in part by pushing against the actin cytoskeleton. Recent studies have suggested that comparable dyneindriven forces may impinge upon the longer microtubules within the axon. Here, we examined a potential role for these forces on axonal retraction and growth cone turning in neurons partially depleted of dynein heavy chain (DHC) by small interfering RNA. While DHC-depleted axons grew at normal rates, they retracted far more robustly in response to donors of nitric oxide than control axons, and their growth cones failed to efficiently turn in response to substrate borders. Live cell imaging of dynamic microtubule tips showed that microtubules in DHC-depleted growth cones were largely confined to the central zone, with very few extending into filopodia. Even under conditions of suppressed microtubule dynamics, DHC depletion impaired the capacity of microtubules to advance into the peripheral zone of the growth cone, indicating a direct role for dynein-driven forces on the distribution of the microtubules. These effects were all reversed by inhibition of myosin-II forces, which are known to underlie the retrograde flow of actin in the growth cone and the contractility of the cortical actin during axonal retraction. Our results are consistent with a model whereby dynein-driven forces enable microtubules to overcome myosin-II-driven forces, both in the axonal shaft and within the growth cone. These dynein-driven forces oppose the tendency of the axon to retract and permit microtubules to advance into the peripheral zone of the growth cone so that they can invade filopodia.

Differential roles of kinesin and dynein in translocation of neurofilaments into axonal neurites

“…In the case of dynein, the actin cortex, like longer MTs, functions as a substrate against which dynein can translocate MTs (and any associated NFs) into and along axonal neurites (Ahmad et al, 1998;Hasaka et al, 2004;Myers et al, 2006;Pfister, 1999;Susalka et al, 2000). The actin cortex also participates in kinesin-mediated anterograde translocation of NFs, both by providing anchorage Francis et al, 2005;Jung et al, 2004;Rao et al, 2002) and perhaps through interactions between kinesin, and/or NFs, and one or more forms of the actin-based motor myosin (Ali et al, 2008;Huang et al, 1999;Jung et al, 2004).…”

Cytoskeletal dynamics in growth-cone steering

“…The exceptions are the looped or highly curved microtubules that are occasionally found in the C-domain (see below). The main mechanism for distal microtubule extension in growth cones is plus-end assembly; there is some evidence for distal (anterograde) polymer translocation, possibly driven by the microtubule motor cytoplasmic dynein (Dent et al, 1999;Zhou et al, 2002;Schaefer et al, 2002;Ma et al, 2004;Myers et al, 2006). Retrograde translocation of microtubules, by contrast, is commonly observed (see below) .…”

“…Similarly, stabilising microtubules in Aplysia growth cones prevents appropriate steering responses on contact with a microbead that is coated with cell-adhesion molecules (Suter et al, 2004). Knocking down the expression of dynein heavy chain using small interfering RNAs suppresses microtubule insertion into filopodia and this is also associated with inhibition of growth-cone turning (Myers et al, 2006) (see also Grabham et al, 2007). By contrast, disassembly of microtubules on one side of the growth cone by an externally applied gradient of the microtubule-depolymerising compound nocodazole causes the growth cone to turn away from that side (Buck and Zheng, 2002).…”

“…When expressed in neurons as GFPfusion proteins, EB1 and EB3 'surf' on the plus ends of growing microtubules in growth cones, appearing as 'comets' with tails that point toward the minus end of the microtubule (Fig. 3) (Morrison et al, 2002;Stepanova et al, 2003;Ma et al, 2004;Myers et al, 2006;Geraldo et al, 2008). EB3-GFP comets can be seen entering filopodia where they usually disappear at some point along their length, presumably because the microtubule has either stopped growing or has depolymerised (Fig.…”

Using fly genetics to dissect the cytoskeletal machinery of neurons during axonal growth and maintenance

“…Other ABPs are involved in indirect crosstalk with MTs by interacting with MTBPs. Reported examples are the ABP drebrin that is linked to the MTBP EB3, the ABP PPP1R9A (better known as spinophilin) that binds to DCX, IQGAP that interacts with CLIP-170, or functional interactions between the actin-binding motor protein myosin II and the MT-associated dynein motor protein complex (Bielas et al, 2007;Geraldo et al, 2008;Myers et al, 2006;Swiech et al, 2011).…”