Live cell imaging of cytoplasmic dynein movement in transfected embryonic rat neurons.

Live cell imaging of the movement of various membrane-bounded organelle cargos has enhanced our understanding of their function. Eukaryotic cells utilize microtubules and two classes of microtubule-based motor proteins, cytoplasmic dynein and members of the kinesin family, to deliver a variety of membrane-bounded organelles and other cargos to their appropriate locations. In order to better understand the functions and regulation of cytoplasmic dynein, we developed a method to study its location and motility in living cells. The technique takes advantage of the long thin axons of cultured hippocampal neurons. We use calcium phosphate to transfect fluorescent-tagged dynein intermediate chain (IC) subunits (DYNC1I) into cultured neurons. When the ICs are expressed at low levels, they are effective probes for the location of the cytoplasmic dynein complex in axons when living cells are imaged with fluorescence microscopy. The fluorescent subunit probes can be used to identify specific cargos of dynein complexes with different IC isoforms as well as the kinetic properties of cytoplasmic dynein.

Live cell imaging reveals differential modifications to cytoplasmic dynein properties by phospho- and dephosphomimic mutations of the intermediate chain 2C S84.

Cytoplasmic dynein is a multisubunit motor protein responsible for intracellular cargo transport toward microtubule minus ends. There are multiple isoforms of the dynein intermediate chain (DYNC1I, IC), which is encoded by two genes. One way to regulate cytoplasmic dynein is by IC phosphorylation. The IC-2C isoform is expressed in all cells, and the functional significance of phosphorylation on IC-2C serine 84 was investigated by using live cell imaging of fluorescent protein-tagged IC-2C wild type (WT) and phospho- and dephosphomimic mutant isoforms in axonal transport model systems. Both mutations modulated dynein functional properties. The dephosphomimic mutant IC-2C S84A had greater colocalization with mitochondria than the IC-2C WT or the phosphomimic mutant IC-2C S84D. The dephosphomimic mutant IC-2C S84A was also more likely to be motile than the phosphomimic mutant IC-2C S84D or the IC-2C WT. In contrast, the phosphomimic mutant IC-2C S84D mutant was more likely to move in the retrograde direction than was the IC-2C S84A mutant. The phosphomimic IC-2C S84D was also as likely as the IC-2C WT to colocalize with mitochondria. Both the S84D phospho- and the S84A dephosphomimic mutants were found to be capable of microtubule minus-end-directed (retrograde) movement in axons. They were also observed to be passively transported in the anterograde direction. These data suggest that the IC-2C S84 has a role in modulating dynein properties.

Trk activation of the ERK1/2 kinase pathway stimulates intermediate chain phosphorylation and recruits cytoplasmic dynein to signaling endosomes for retrograde axonal transport.

The retrograde transport of Trk-containing endosomes from the axon to the cell body by cytoplasmic dynein is necessary for axonal and neuronal survival. We investigated the recruitment of dynein to signaling endosomes in rat embryonic neurons and PC12 cells. We identified a novel phosphoserine on the dynein intermediate chains (ICs), and we observed a time-dependent neurotrophin-stimulated increase in intermediate chain phosphorylation on this site in both cell types. Pharmacological studies, overexpression of constitutively active MAP kinase kinase, and an in vitro assay with recombinant proteins demonstrated that the intermediate chains are phosphorylated by the MAP kinase ERK1/2, extracellular signal-regulated kinase, a major downstream effector of Trk. Live cell imaging with fluorescently tagged IC mutants demonstrated that the dephosphomimic mutants had significantly reduced colocalization with Trk and Rab7, but not a mitochondrial marker. The phosphorylated intermediate chains were enriched on immunoaffinity-purified Trk-containing organelles. Inhibition of ERK reduced the amount of phospho-IC and the total amount of dynein that copurified with the signaling endosomes. In addition, inhibition of ERK1/2 reduced the motility of Rab7- and TrkB-containing endosomes and the extent of their colocalization with dynein in axons. NGF-dependent survival of sympathetic neurons was significantly reduced by the overexpression of the dephosphomimic mutant IC-1B-S80A, but not WT IC-1B, further demonstrating the functional significance of phosphorylation on this site. These results demonstrate that neurotrophin binding to Trk initiates the recruitment of cytoplasmic dynein to signaling endosomes through ERK1/2 phosphorylation of intermediate chains for their subsequent retrograde transport in axons.