Insights on UNC‐104‐dynein/dynactin interactions and their implications on axonal transport in Caenorhabditis elegans

Abstract

Bidirectional cargo transport in neurons can be explained by two models: the "tug-ofwar model" for short-range transport, in which several kinesin and dynein motors are bound to the same cargo but travel in opposing directions, and by the "motor coordination model" for long-range transport, in which small adaptors or the cargo itself activates or deactivates opposing motors. Direct interactions between the major axonal transporter kinesin-3 UNC-104(KIF1A) and the dynein/dynactin complex remains unknown. In this study, we dissected and evaluated the interaction sites between UNC-104 and dynein as well as between UNC-104 and dynactin using yeast two-hybrid assays. We found that the DYLT-1(Tctex) subunit of dynein binds near the coiled coil 3 (CC3) of UNC-104, and that the DYRB-1(Roadblock) subunit binds near the CC2 region of UNC-104. Regarding dynactin, we specifically revealed strong interactions between DNC-6(p27) and the FHA-CC3 stretch of UNC-104, as well as between the DNC-5(p25) and the CC2-CC3 region of UNC-104. Motility analysis of motors and cargo in the nervous system of Caenorhabditis elegans revealed impaired transport of UNC-104 and synaptic vesicles in dynein and dynactin mutants (or in RNAi knockdown animals). Further, in these mutants UNC-104 clustering along axons was diminished. Interestingly, when dynamic UNC-104 motors enter a stationary UNC-104 cluster their dwelling times are increased in dynein mutants (suggesting that dynein may act as an UNC-104 activator). In summary, we provide novel insights on how UNC-104 interacts with the dynein/dynactin complex and how UNC-104 driven axonal transport depends on dynein/dynactin in C. elegans neurons. K E Y W O R D Saxonal transport, Caenorhabditis elegans, KIF1A, kinesin, molecular motors, yeast two-hybrid

Characterization of TAG‐63 and its role on axonal transport in C. elegans

“…Nevertheless, antibody staining after tag‐63 knockdown was significantly reduced (Figure D + E). Note that we have previously shown the efficiency of RNAi in neuronal tissues without the need of cross‐breeding with RNAi sensitive strains, and also for this study we provide data reflecting the efficiency of tag‐63 knockdown in neurons (Figure S8A + B). Though we have no information regarding the epitope sequence of the NFH200 antibody, we obtained the immunogen sequence for the NEFH antibody, and our analysis revealed that this antibody may cross‐react with TAG‐63, but likely not with IFP‐1, IFB‐1, or IFA‐4 (Figure S3B).…”

“…As the disintegration of both vimentin and NFs are known to affect MT‐based transport, we analyzed the motility of kinesin‐3 UNC‐104 and its cargo SNB‐1 (synaptobrevin‐1) in ALM neurons of wild type, TAG‐63 knockout as well as TAG‐63 knockdown (RNAi) worms. Figure shows anterograde and retrograde velocity profiles of motor UNC‐104 as well as of its cargo SNB‐1, both tagged with mRFP (revealed from separate strains and experiments). Anterograde movements of UNC‐104 are reduced in tag‐63 ( ok471 ) animals (Figure A + D) pointing toward a regulatory role of TAG‐63 on UNC‐104's motor activity.…”

PTP-3(LAR PTPR) promotes intramolecular folding of SYD-2(liprin-α) to inactivate UNC-104(KIF1A) in neurons

“…6B). However, it needs to be noted that UNC-104 may exist in complexes with dynein [12] or that multiple motors of different directionalities (antagonistic motors) may accumulate and bind to the same cargo vesicle resulting in saltatory movements of that vesicle. Because we only observe the movements of fluorescent UNC-104 particles, we may expect that we measure either moving parameters of single motors or those of multiple motors accumulated on a single vesicle.…”

“…2) Cargo binding activates kinesins [11]. 3) Physical pulling via opposing motor dynein activates kinesins [12]. 4) Binding of small adaptor proteins such as SYD-2 and LIN-2 activates kinesin [2,3].…”

Molecular mechanisms governing axonal transport: a C. elegans perspective

“…This is consistent with the "tug-of-war" model proposed in in vitro and cell culture studies (Hancock, 2018). Conversely, UNC-104 has also been reported to physically interact with several components of the Dynein-Dynactin complex, such as DYLT-1, DYRB-1, DNC-5 and DNC-6, which have been shown to affect the velocity and dwell times of UNC-104 along the neuronal process and the anterograde transport of SNB-1 in C. elegans neurons (Chen et al, 2019). It has further been proposed that these interactions are necessary to activate UNC-104, promote anterograde axonal transport (Chen et al, 2019), and likely function to localize components of the retrograde transport machinery to distal tips of neuronal processes (Figure 2(a)).…”