Abstract
Cytoplasmic dynein is the primary minus-end-directed microtubule motor protein in animal cells, performing a wide range of motile activities, including transport of vesicular cargos, mRNAs, viruses, and proteins. Lissencephaly-1 (LIS1) is a highly conserved dynein-regulatory factor that binds directly to the dynein motor domain, uncoupling the enzymatic and mechanical cycles of the motor and stalling dynein on the microtubule track. Dynactin, another ubiquitous dynein-regulatory factor, releases dynein from an autoinhibited state, leading to a dramatic increase in fast, processive dynein motility. How these opposing activities are integrated to control dynein motility is unknown. Here, we used fluorescence single-molecule microscopy to study the interaction of LIS1 with the processive dynein-dynactin-BicD2N (DDB) complex. Surprisingly, in contrast to the prevailing model for LIS1 function established in the context of dynein alone, we found that binding of LIS1 to DDB does not strongly disrupt processive motility. Motile DDB complexes bound up to two LIS1 dimers, and mutational analysis suggested that LIS1 binds directly to the dynein motor domains during DDB movement. Interestingly, LIS1 enhanced DDB velocity in a concentration-dependent manner, in contrast to observations of the effect of LIS1 on the motility of isolated dynein. Thus, LIS1 exerts concentration-dependent effects on dynein motility and can synergize with dynactin to enhance processive dynein movement. Our results suggest that the effect of LIS1 on dynein motility depends on both LIS1 concentration and the presence of other regulatory factors such as dynactin and may provide new insights into the mechanism of LIS1 haploinsufficiency in the neurodevelopmental disorder lissencephaly.
Highlights
Cytoplasmic dynein is the primary minus-end– directed microtubule motor protein in animal cells, performing a wide range of motile activities, including transport of vesicular cargos, mRNAs, viruses, and proteins
Our results suggest that the effect of LIS1 on dynein motility depends on both LIS1 concentration and the presence of other regulatory factors such as dynactin and may provide new insights into the mechanism of LIS1 haploinsufficiency in the neurodevelopmental disorder lissencephaly
When we mixed 15 nM purified, SNAP-647-labeled LIS1 with ϳ5 nM DDB, we readily observed LIS1 bound to both processively moving and diffusing DDB complexes (Fig. 1B and supplemental Movie S1). We found that this concentration of LIS1 provided an optimal signal-to-noise ratio in our assays, because higher LIS1 concentrations led to high background that obscured the LIS1 signal on the MTs, and lower concentrations led to few LIS1 molecules bound to DDB
Summary
To directly observe the effects of LIS1 on DDB motility, we utilized multicolor single molecule total internal reflection fluorescence (TIRF) microscopy. When we mixed 15 nM purified, SNAP-647-labeled LIS1 with ϳ5 nM DDB, we readily observed LIS1 bound to both processively moving and diffusing DDB complexes (Fig. 1B and supplemental Movie S1). We observed rare dynamic events where single DDB-L complexes bound differentially labeled LIS1 molecules at different times during processive or diffusive movement (Fig. 3B), further suggesting that most DDB molecules bind single LIS1 dimers in our conditions. The total fraction of complexes found in each velocity population was not substantially changed by LIS1 addition, indicating that LIS1 affects the velocity of both slower and faster complexes (Fig. 4E) These results show that LIS1 modulates DDB velocity through concentration-dependent changes in the binding occupancy of dynein motor domains. High levels of LIS1 facilitate faster DDB velocities at a modest expense of total numbers of processive complexes
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