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Molecular motors often work in teams to move a cellular cargo. Yet measuring the forces exerted by each motor is challenging. Using a sensor made with denatured ssDNA and multi-color fluorescence, we measured picoNewtons of forces and nanometer distances exerted by individual constrained kinesin-1 motors acting together while driving a common microtubule . We find that kinesins primarily exerted less than 1 pN force, even while the microtubule is bypassing artificial obstacles of 20-100 nanometer size. Occasionally, individual forces increase upon encountering obstacles, although at other times they do not, with the cargo continuing in a directional manner. Our high-throughput technique, which can measure forces by many motors simultaneously, is expected to be useful for many different types of molecular motors.
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http://dx.doi.org/10.1039/d2nr01701f | DOI Listing |
Kinesin-1 is a robust motor that carries intracellular cargos towards the plus ends of microtubules. However, optical trapping studies reported that kinesin-1 is a slippery motor that quickly detaches from the microtubule, and multiple kinesins are incapable of teaming up to generate large collective forces. This may be due to the vertical (z) forces that the motor experiences in a single bead trapping assay, accelerating the detachment of the motor from a microtubule.
View Article and Find Full Text PDFBiophys J
September 2025
Department of Molecular Physiology and Biophysics, University of Vermont Larner College of Medicine, Burlington, VT 05405. Electronic address:
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 in vitro? 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%).
View Article and Find Full Text PDFbioRxiv
August 2025
Department of Cell & Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA. 48109.
Microtubules are cytoskeletal filaments with critical roles in cell division, cell motility, intracellular trafficking, and cilium function. In cells, subsets of microtubules are selectively marked by posttranslational modifications (PTMs), which control the ability of microtubule-associated proteins (MAPs) and molecular motors to engage microtubules. Detyrosination (ΔY) and ΔC2 are PTMs of α-tubulin, wherein one or two residues, respectively, are enzymatically removed from the C-terminus of the protein.
View Article and Find Full Text PDFJ Cell Sci
September 2025
Section of Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.
Xenopus egg extracts can self-organize into cell-like compartments without the classic microtubule organizer centrosome. Compartment formation requires microtubules, but the organization of microtubules throughout the process remains unclear. Here, we show that the earliest organized microtubule structures to emerge during cell-like compartment formation are centrosome-independent asters.
View Article and Find Full Text PDFCytoskeleton (Hoboken)
August 2025
Department of Life Sciences Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan.
Kinesin-1 is a dimeric motor protein that moves towards the microtubule plus-end in a hand-over-hand fashion. However, the minimal motor domain of kinesin-1 is a single head, and the mechanism by which minimal motor domains generate the force for directional movement remains poorly understood. Here, we engineered artificial tethers (polyethylene glycol, single-stranded DNA, or double-stranded DNA) within the motor domain to investigate whether tether properties such as charge, length, and stiffness affect the motility of teams of kinesin-1 monomers.
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