In vivo optical trapping indicates kinesin’s stall force is reduced by dynein during intracellular transport

Abstract

Kinesin and dynein are fundamental components of intracellular transport, but their interactions when simultaneously present on cargos are unknown. We built an optical trap that can be calibrated in vivo during data acquisition for each individual cargo to measure forces in living cells. Comparing directional stall forces in vivo and in vitro, we found evidence that cytoplasmic dynein is active during minus- and plus-end directed motion, whereas kinesin is only active in the plus direction. In vivo, we found outward (∼plus-end) stall forces range from 2 to 7 pN, which is significantly less than the 5- to 7-pN stall force measured in vitro for single kinesin molecules. In vitro measurements on beads with kinesin-1 and dynein bound revealed a similar distribution, implying that an interaction between opposite polarity motors causes this difference. Finally, inward (∼minus-end) stalls in vivo were 2–3 pN, which is higher than the 1.1-pN stall force of a single dynein, implying multiple active dynein.

Motor Domain Phosphorylation Modulates Kinesin-1 Transport

“…Even in the absence of an autoinhibitory tail domain, the S175D kinesin-and dynein-coated beads demonstrated a bias toward minus-end transport. A recent study indicated that dynein remains bound to the microtubule during plus-end transport, dragging behind kinesin (27). Dynein dragging acts as an opposing force on the order of a few pN that kinesin must work against.…”

“…Optical Trapping Assay-The optical trap setup was used as described previously with the addition of a force-feedback loop to maintain a constant load for force-velocity curve measurements (27). Motors were nonspecifically attached to carboxylated beads in a manner similar to that used in fluorescence imaging experiments.…”

Force measurements with optical tweezers inside living cells

“…We focused on the A549 cell line as prior experiments using a variety of calibration approaches had shown discrepant stall force measurement of kinesin and dynein when compared to in vitro assays 16,17 . Following the procedures in these references, we identified lipid droplets moving in opposite directions, labeling them "inward" and "outward" depending on whether they were moving from the cell periphery towards the cell nucleus or vice versa, respectively.…”

Perspective: Reaches of chemical physics in biology

“…80,81 In the near future, such simulations will scale up from procaryotic to eucaryotic cells. Awaiting whole cell simulations is a new generation of experiments that look at protein folding in different organelles of the cell, 82 super-resolution structure of the cell such as the recently discovered "skeleton" of neurons 83 or time-resolved super-resolution dynamics in cell membranes, 84 in vivo characterization of motors and other cellular machinery, 85 and cryoelectron microscopy of cellular machinery. Many areas, from neurobiology to developmental biology will benefit from the development of these tools.…”