TI - Results . AB - We generated a stable PC12 cell line expressing low levels ( GFP-tagged IC isoform 2C ( GFP-IC-2C ) and used it to characterize the properties of dynein complexes containing the IC-2C isoform . In cell bodies , few discrete GFP-IC-containing structures were resolved due to the presence of a large soluble dynein pool . However , in neurites , the distribution of GFP-dynein was punctate and comparable to that observed with an antibody to dynein ( Fig 1 ) . Some regions of the neurites were wide enough to distinguish microtubules , and in these regions , the GFP-dynein puncta colocalized with the microtubules ( Fig 1 ) . Fig. 2 A shows that most of the endogenous dynein IC , and the GFP-IC , copelleted with microtubules . This indicated that the GFP-IC , like the endogenous IC , was bound to the microtubule-binding motor domain of the dynein complex , the heavy chain . In addition , dynein containing the GFP-IC was released from microtubules in the presence of Mg-ATP , demonstrating that the GFP-IC containing dynein hydrolyzed ATP . Fig. 2 B shows that after sucrose density gradient sedimentation , all of the GFP-IC-2C , like the endogenous IC , copurified exclusively with 20S dynein complexes near the bottom of the gradient ( Fig 2 B ) . The absence of GFP-IC at the top of the gradient showed that there was no free pool of GFP-IC . Therefore all the GFP-IC-2C incorporated into functional dynein complexes , and the GFP-IC puncta observed in cells corresponded to these complexes . In addition , two membrane vesicle markers , synaptophysin and the transmembrane neurotrophin receptor TrkA , were purified by immunoaffinity chromatography from a membrane fraction by antibodies to GFP coupled to magnetic beads ( Fig 2 C ; and see Fig 8 C ) . This result established that dynein containing the GFP-IC associated with membranous organelles . In living PC12 cell neurites , GFP-IC-2C dynein puncta were observed to move rapidly along linear tracks in both the anterograde ( toward the tip ) and retrograde ( toward the cell body ) directions ( Fig. 3 A and Video 1 , available at . http : . . . . . . . . . . . . . . /www.jcb.org/cgi/content/full/jcb. lt @@@@@ gt 200803150/DC ) . We found that 92% of the growing microtubules ( n = 53 ) in PC12 cells were oriented with their plus ends toward the tip by use of the EB3 polarity assay . Therefore , as in axons , anterograde movement in PC12 cell neurites is directed toward microtubule plus ends , and retrograde movement is directed toward microtubule minus ends . Frame-to-frame tracking of the movement of individual dynein puncta showed that their velocity changed during excursions ( see Materials and methods ; Fig 3 B ) . The interval velocities of the puncta were in the range of 0.3-3.1 mum/s , and the mean interval velocity for retrograde dynein movement was 0.9 mum/s ( Fig 3 C and Table I ) . A larger number of the dynein puncta were observed moving in the anterograde direction than the retrograde direction in the PC12 cell neurites . This was consistent with the observation that dynein accumulated in growth cones . To further demonstrate that the retrograde motility of GFP-IC dynein complexes was the result of their motor activity , siRNA specific for the untranslated region of IC-2 gene products was used to deplete the endogenous dynein pool while allowing expression of the GFP-tagged IC ( Fig 4 ) . Robust GFP-dynein puncta movement in PC12 cell neurites continued after the depletion of the endogenous IC-2 isoforms , which would normally cause the cessation of movement ( Fig 4 and Video 2 ) . This observation confirmed that the GFP-dynein complexes moving in the retrograde direction were active motors , rather than cargo of the endogenous dynein . To identify differences in the functional roles of the dynein complexes with distinct ICs in neurons , we used live cell imaging of fluorescent dynein IC isoforms transfected into cultured hippocampal neurons . The kinetic and cargo-binding properties of dynein complexes with two isoforms , the ubiquitous isoform IC-2C and the neuron-specific isoform IC-1B , were determined . In the axons of hippocampal neurons , dynein complexes containing IC-2C and IC-1B moved rapidly in both the anterograde and retrograde directions ( Fig. 5 , A and B ; and Videos 3 and 4 , available at . http : . . . . . . . . . . . . . . /www.jcb.org/cgi/content/full/jcb. lt @@@@@ gt 200803150/DC ) . In contrast to the movement of IC-2C-containing dynein complexes in PC12 cell neurites , no directional bias for either of the two distinct dynein complexes was observed in hippocampal neuron axons . Significantly more of the dynein complexes containing IC-2C were found to be nonmotile than the dynein complexes containing IC-1B ( P lt 00005 ; Table II ) . Large numbers of both classes of dynein complexes displayed jiggling motion but no net motility . Occasionally , the jiggling movement was observed to precede or follow a motile excursion . There was no significant difference between the retrograde interval velocity distributions of the two classes of dynein ( Fig 6 ) . The range of velocities observed for dynein complexes with either IC-2C or IC-1B was 0.3-3.6 mum/s in hippocampal axons . However , compared with PC12 cells , a larger number of slow velocities were observed in axons . This accounts for the net slower mean interval velocities ( 07 mum/s ) calculated for both dynein isoforms . The kinetics of dynein excursions in the retrograde direction were also analyzed . There was no significant difference in either the mean excursion velocities or run lengths when dynein complexes containing IC-2C and IC-1B were compared . However , on average , the excursions of dynein containing IC-1B lasted ~30% longer ( P lt 00003 ) . Some puncta from both classes of dynein were observed to switch direction during excursive movement in hippocampal axons . Interestingly , a dynein puncta was as likely to switch from the retrograde to the anterograde direction as the anterograde to retrograde direction ( unpublished data ) . No significant difference was observed between the anterograde interval velocity distributions of dynein complexes with IC-1B and IC-2C in axons ( Table III and not depicted ) . However , the mean anterograde excursion velocity of dynein with IC-2C was slightly faster than that of dynein with IC-1B ( P lt 002 ) . We also characterized the binding of dynein complexes with different ICs to one cargo , TrkB signaling endosomes . In neurons , the retrograde transport of signaling endosomes ( which contain activated , neurotrophin-binding Trks ) to the nucleus is essential for axonal survival . Cortical and hippocampal neurons express the brain-derived neurotrophic factor receptor TrkB . We have shown that hippocampal neurons express both the IC-2C ( ubiquitous ) and IC-1B ( neuron-specific ) IC isoforms . When the distribution of TrkB was compared with that of the two different dynein complexes in living hippocampal neurons , a significant difference in the extent of colocalization was observed ( P lt 0005 ; Fig 7 A ) . Only 2.6 + - 1.5% of the IC-2C spots colocalized with TrkB , whereas 17.8 + - 4.6% of the IC-1B colocalized with TrkB . Thus , dynein containing IC-1B was much more likely to colocalize with TrkB carrier vesicles than dynein containing IC-2C . Significantly , coordinate movement of TrkB with dynein containing IC-1B was also observed , demonstrating that dynein with the IC-1B isoform is responsible for the retrograde transport of TrkB containing endosomes ( Fig. 7 B and Video 5 , available at . http : . . . . . . . . . . . . . . /www.jcb.org/cgi/content/full/jcb. lt @@@@@ gt 200803150/DC ) . In contrast , transport of TrkB containing endosomes by dynein containing IC-2C was not found in axons . To confirm that TrkB did not associate with IC-2C dynein complexes , signaling endosomes were immunopurified from the cortex , a brain region with high levels of TrkB expression , by using magnetic beads coupled with anti-Trk antibodies . Antibodies that distinguish between IC isoforms were used to demonstrate that dynein complexes containing IC-1 copurified with the Trk containing vesicles , but dynein containing IC-2 did not ( Fig 7 C ) . Thus , TrkB vesicles from cortical neurons were associated with IC-1 but were not associated with any IC-2 isoform . Together , the live cell imaging and biochemical data demonstrate that dynein complexes containing IC-1B but not dynein containing IC-2C are responsible for the transport of TrkB containing signaling endosomes in cortical and hippocampal neurons . PC12 cells express the NGF receptor TrkA . Because PC12 cells do not express IC-1 isoforms , we tested the hypothesis that IC-2C-containing dynein was involved in the transport of TrkA signaling endosomes in these cells . We compared the distribution of GFP-IC-2C-containing dynein complexes and TrkA in PC12 cell neurites . After IC-2-specific siRNA was used to deplete the endogenous IC-2 pool , we found significant overlap in the distributions of dynein and TrkA in PC12 cell neurites ( Fig 8 A ) . NGF binding to the extracellular domain of TrkA leads to autoPHOSphorylation of its cytoplasmic tail , and the activated Trk is internalized with bound NGF in signaling endosomes . Dynein also colocalized with activated ( PHOSphorylated ) TrkA in neurites ( Fig 8 B ) . To confirm the binding of GFP-IC-2C-containing dynein to TrkA , signaling endosomes were immunopurified using antibodies to GFP bound to magnetic beads . TrkA was found to coimmunoprecipitate with the GFP-IC-2C dynein ( Fig 8 C ) . We next sought to determine if the addition of NGF to PC12 cells modulated the interaction of dynein with TrkA . Membrane-bounded organelles from control and NGF -stimulated PC12 cells were isolated by immunoaffinity purification with antibodies to dynein on magnetic beads . More activated TrkA copurified with dynein from NGF -stimulated cells than from control cells (Fig 9 A) . This demonstrated that the formed signaling endosomes recruited dynein for retrograde transport . We previously found that the addition of NGF increased the PHOSphorylation of PC12 cell ICs . We therefore investigated whether the addition of NGF regulated the kinetic properties of dynein containing GFP-IC-2C in the stable PC12 cell line . The addition of NGF significantly increased the mean interval velocity of the dynein puncta moving in the retrograde direction by ~30% , from 0.9 to 1.2 mum/s ( P lt 000017 ) , but there was no change in the anterograde interval velocity ( Fig 9 B and Table I ) . Also , there was a significant increase in the relative amount of dynein puncta moving in the retrograde direction , an increase from 34 to 52% ( P lt 00002 ) upon the addition of NGF ( Fig 9 B and Table I ) . These data indicate that NGF stimulation of PC12 cells leads to increased association of activated TrkA containing organelles with dynein and increased dynein retrograde motor activity .