Dynein mediates retrograde neurofilament transport within axons and anterograde delivery of NFs from perikarya into axons: Regulation by multiple phosphorylation events

Abstract

We examined the respective roles of dynein and kinesin in axonal transport of neurofilaments (NFs). Differentiated NB2a/d1 cells were transfected with green fluorescent protein-NF-M (GFP-M) and dynein function was inhibited by co-transfection with a construct expressing myc-tagged dynamitin, or by intracellular delivery of purified dynamitin and two antibodies against dynein's cargo domain. Monitoring of the bulk distribution of GFP signal within axonal neurites, recovery of GFP signal within photobleached regions, and real-time monitoring of individual NFs/punctate structures each revealed that pertubation of dynein function inhibited retrograde transport and accelerated anterograde, confirming that dynein mediated retrograde axonal transport, while intracellular delivery of two anti-kinesin antibodies selectively inhibited NF anterograde transport. In addition, dynamitin overexpression inhibited the initial translocation of newly-expressed NFs out of perikarya and into neurites, indicating that dynein participated in the initial anterograde delivery of NFs into neurites. Delivery of NFs to the axon hillock inner plasma membrane surface, and their subsequent translocation into neurites, was also prevented by vinblastine-mediated inhibition of microtubule assembly. These data collectively suggest that some NFs enter axons as cargo of microtubues that are themselves undergoing transport into axons via dynein-mediated interactions with the actin cortex and/or larger microtubules. C-terminal NF phosphorylation regulates motor association, since anti-dynein selectively coprecipitated extensively phosphorylated NFs, while anti-kinesin selectively coprecipitated less phosphorylated NFs. In addition, however, the MAP kinase inhibitor PD98059 also inhibited transport of a constitutively-phosphorylated NF construct, indicating that one or more additional, non-NF phosphorylation events also regulated NF association with dynein or kinesin.

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

“…Introduction of haptenized exogenous NF fusion proteins into neurons has facilitated analyses of NF axonal transport. NF subunits translocate within axons in multiple forms, including as short NFs, non-filamentous punctate structures ('puncta') and diffuse material that might represent unassembled subunits or hetero-oligomeric subunit assemblies (Jung et al, 1998;Motil et al, 2006;Roy et al, 2000;Prahlad et al, 2000;Theiss et al, 2005;Wang et al, 2000;Yabe et al, 1999;Yabe et al, 2001a;Yabe et al, 2001b;Yuan et al, 2009). The interplay, if any, among these various forms remains unresolved.…”

“…Transport of NFs into and along axons requires an intact MT system Jung et al, 1998;Jung et al, 2004;Yabe et al, 2000) and the action of the MT motor proteins kinesin and dynein (He et al, 2005;Motil et al, 2006;Prahlad et al, 2000;Shah et al, 2000;Theiss et al, 2005;Uchida et al, 2009;Xia et al, 2003;Yabe et al, 1999;Yabe et al, 2000). As with other axonal constituents, kinesin mediates anterograde NF transport (Prahlad et al, 2000;Theiss et al, 2005;Xia et al, 2003;Yabe et al, 1999;Yabe et al, 2000), whereas dynein mediates retrograde transport (He et al, 2005;Motil et al, 2006;Shah et al, 2000;Theiss et al, 2005).…”

“…As with other axonal constituents, kinesin mediates anterograde NF transport (Prahlad et al, 2000;Theiss et al, 2005;Xia et al, 2003;Yabe et al, 1999;Yabe et al, 2000), whereas dynein mediates retrograde transport (He et al, 2005;Motil et al, 2006;Shah et al, 2000;Theiss et al, 2005). However, the dynamics of assembly and transport of NFs are complex; these MT-based motor proteins have multiple interactions with actin and the actin-based motor myosin Jung et al, 2004;Rao et al, 2002).…”

“…However, the dynamics of assembly and transport of NFs are complex; these MT-based motor proteins have multiple interactions with actin and the actin-based motor myosin Jung et al, 2004;Rao et al, 2002). Unexpectedly, the retrograde-directed motor dynein also participates in the anterograde delivery of NF forms into axonal neurites (Motil et al, 2006). The probable mechanism underlying this initially counterintuitive role of dynein is the unique role of this motor protein in delivery of MTs into axons; as dynein can associate with MTs through its cargo domain, as well as its motor domain, it is capable of translocating shorter MTs in an anterograde direction through cargo-based interactions with relatively larger MTs and/or actin filaments.…”

Intermediate Filaments as a Target of Signaling Mechanisms in Neurotoxicity

“…The carboxyl-terminal phosphorylation of NF-H progressively restricts association of NFs with kinesin, the axonal anterograde motor protein and stimulates its interaction with dynein, the axonal retrograde motor protein [63]. This event could represent one of the mechanisms by which carboxyl-terminal phosphorylation would slow NF axonal transport.…”