Bensel Brandon M, Previs Samantha B, Fagnant Patricia M, Trybus Kathleen M, Walcott Sam, Warshaw David M
Department of Molecular Physiology and Biophysics, University of Vermont Larner College of Medicine, Burlington, VT 05405.
Department of Mathematical Sciences, and Bioinformatics and Computational Biology, Worcester Polytechnic Institute, Worcester, MA 01609.
bioRxiv. 2025 Jun 3:2025.05.06.652443. doi: 10.1101/2025.05.06.652443.
Intracellular vesicular transport by kinesin-1 motors through numerous 3-dimensional (3D) microtubule (MT) intersections must be regulated to support proper vesicle delivery. Knowing kinesin-1 can be regulated via autoinhibition, does kinesin-1 exhibit autoinhibition on cargo, and could this regulate vesicular transport through 3D MT intersections ? To answer this question, we compared liposome transport by ~10 nearly full-length kinesin-1 motors with KLC bound (KinΔC) versus constitutively active control (K543). In 3D MT intersections, KinΔC-liposomes terminate (48%), go straight (43%), but rarely turn (9%), starkly contrasting K543-liposomes which go straight (57%), turn (31%), but rarely terminate (12%). On single MTs, KinΔC-liposomes have reduced run lengths and detachment forces versus K543-liposomes, suggesting autoinhibition reduces MT engagement, as supported by 3-fold lower KinΔC MT landing rates versus K543, and mechanistic modeling. Furthermore, kinesore, a small molecule that overcomes kinesin-1 autoinhibition, restores KinΔC's MT engagement. Thus, we propose that partial kinesin-1 autoinhibition while cargo-bound may fine-tune cargo delivery to support physiological demands.
驱动蛋白-1马达通过众多三维（3D）微管（MT）交叉点进行的细胞内囊泡运输必须受到调控，以支持囊泡的正确递送。已知驱动蛋白-1可通过自身抑制进行调控，那么驱动蛋白-1在结合货物时是否表现出自身抑制作用，以及这是否会调节通过3D MT交叉点的囊泡运输呢？为了回答这个问题，我们比较了约10个与KLC结合的几乎全长驱动蛋白-1马达（KinΔC）和组成型活性对照（K543）对脂质体的运输情况。在3D MT交叉点处，KinΔC-脂质体终止运输的比例为48%，直线运输的比例为43%，但很少转向（9%），这与K543-脂质体形成鲜明对比，后者直线运输的比例为57%，转向的比例为31%，但很少终止运输（12%）。在单根MT上，与K543-脂质体相比，KinΔC-脂质体的运行长度和脱离力降低，这表明自身抑制降低了与MT的结合，3倍低于K543的KinΔC MT着陆率以及机理模型也支持了这一点。此外，一种克服驱动蛋白-1自身抑制的小分子kinesore可恢复KinΔC与MT的结合。因此，我们提出，在结合货物时，驱动蛋白-1的部分自身抑制可能会微调货物递送以支持生理需求。
