TI - Discussion . AB - Cytoplasmic dynein is the motor for microtubule minus end-directed ( retrograde ) transport . Because all cytoplasmic dynein complexes contain the same motor domain , it has been hypothesized that the isoforms of the other subunits were involved in linking specific cargoes to the dynein complex and for specific regulation of the dynein motor activity . Supporting this hypothesis , we have shown that dynein complexes that contain the neuron-specific IC IC-1B bind and transport TrkB containing endosomes . In contrast , dynein complexes containing the ubiquitous IC IC-2C did not associate with or transport the TrkB signaling endosomes . Thus , although neurons express several IC isoforms , only dynein complexes with a specific IC ( IC-1B ) were used in the transport of TrkB . Interestingly , almost 20% of the IC-1B-containing dynein was used for signaling endosome transport . This is a relatively large amount to be used for the retrograde transport of a single class of organelle , especially considering that the pool includes dynein being transported in the anterograde direction . In contrast , in PC12 cells , the highly related receptor tyrosine kinase , TrkA , bound to and was transported by dynein complexes containing the IC-2C IC . Together these data indicate that neuronal-specific dynein isoforms are used in neurons for transporting particular critical cargoes . The mechanism by which the ICs specify the distinct dynein complexes that associate with the different Trk signaling endosomes remains to be determined . It has been suggested that one of the cytoplasmic dynein light chains , DYNLT1 ( previously called Tctex1 ) , binds to all three Trk growth factor receptors . However , we have found that the DYNLT1 light chain binds to all six ICs . Thus , DYNLT1 does not provide the specificity necessary to account for our findings . The observation that kinase -dead TrkB-GFP is not transported toward the cell body suggests that Trk kinase activity is important for dynein transport . One function of the alternative splicing of the ICs may be to generate unique PHOSphorylation sites to regulate dynein activity ( Vaughan and Vallee , 1995 ) . It is tempting to speculate that IC PHOSphorylation by a downstream effector of the Trk kinases also leads to the recruitment of the appropriate dynein isoform . This hypothesis is consistent with our observation that NGF , which binds to the TrkA receptor , stimulates increased IC PHOSphorylation and , as shown here , increased retrograde dynein activity in PC12 cells . Dynein complexes with GFP-ICs displayed ATP-sensitive microtubule binding and copurified with membrane-bounded organelles . We further found that the copurification of dynein complexes and Trks required the presence of a membranous organelle ( unpublished data ) . In addition , we demonstrated specific differences in the motor activities of dynein complexes containing the distinct IC isoforms . When the kinetics of retrograde dynein motor complexes containing IC-2C and IC-1B were compared , we found that IC-2C-containing dynein complexes were less likely to be motile . Although the two complexes had identical interval velocity distributions , the excursions of IC-1B-containing dynein complexes were , on average , of longer duration . These data suggest that the different ICs isoforms do modulate dynein motor activity . They are also consistent with the recent finding that the phenotype of dynein motor mutations in the Caenorhabditis elegans were suppressed by the deletion of specific cargo-binding subunit isoforms . Together , the data support the hypothesis that the subunit isoforms differentially regulate dynein activity . In contrast to our observations of IC-2C in PC12 cells , there was no significant difference in the number of IC-2C and IC-1B dynein puncta moving in the anterograde and retrograde directions in axons . The neuronal dynein puncta were observed to switch directions but there was no preference for a specific change in direction for either dynein variant . When we compared the anterograde kinetics of dynein complexes with the two distinct IC isoforms in cultured embryonic hippocampal axons , no difference was observed in the interval velocity distribution of the two dynein complexes . However , a small but statistically significant difference ( P lt 002 ; Table III ) was observed in the mean anterograde excursion velocities ; dynein with IC-1B moved slower in cultured hippocampal neurons . We previously found that in adult rat optic nerves , dynein with IC-2C was transported in the anterograde fast transport , whereas dynein complexes with IC-1B were transported in the anterograde slow component b . Transport of IC-2C dynein complexes in the anterograde fast component suggested that they were transported as cargo by a member of the kinesin family . Consistent with this , an overlap in the distributions of GFP-IC-2C and Kinesin-1 was observed when monomeric red fluorescent protein ( mRFP ) -Kinesin-1 ( Kif5a ) was transfected into PC12 cells and Kinesin-1 was present when organelles were purified from the GFP-IC-2C cell line by immunoaffinity purification with antibodies to GFP ( unpublished data ) . Although the anterograde kinetics of IC-1B dynein observed in cultured embryonic hippocampal neurons were more rapid than those observed in the axons of adult optic nerve , it is notable that our results are consistent with recent observations of the transport of slow component b proteins in hippocampal neurons synuclein and two other slow component b proteins were transported at velocities comparable those of dynein containing IC-1B . Other isoforms of dynein subunits have been implicated in linking dynein to specific cargo ; e.g. , light IC 1 (DYNC1LI1) binds to pericentrin and DYNLT1 binds to rhodopsin , whereas DYNLT3 binds to Bub3 . The IC subunit is involved in binding dynein to cargoes through its interaction with the p150 subunit of dynactin ( Karki and Holzbaur , 1995 ; Vaughan and Vallee , 1995 ) . The IC also binds some cargoes directly . The results presented here demonstrate for the first time that specific dynein variants defined by different ICs isoforms are responsible for the transport of specific membrane-bounded organelles . Interestingly , although the chromaffin-derived PC12 cells make use of the ubiquitous IC isoform IC-2C for Trk signaling endosome transport , neurons that express IC-2C use a different IC isoform for Trk transport , one which is expressed exclusively in neurons . Therefore it will be important to identify and compare the regulatory pathways in the different cell types to understand the mechanisms involved in the recruitment of the different dynein complexes to signaling endosomes .