Force measurements on cargoes in living cells reveal collective dynamics of microtubule motors

Abstract

Many cellular cargoes move bidirectionally along microtubules, driven by teams of plus-and minus-end-directed motor proteins. To probe the forces exerted on cargoes during intracellular transport, we examined latex beads phagocytosed into living mammalian macrophages. These latex bead compartments (LBCs) are encased in membrane and transported along the cytoskeleton by a complement of endogenous kinesin-1, kinesin-2, and dynein motors. The size and refractive index of LBCs makes them well-suited for manipulation with an optical trap. We developed methods that provide in situ calibration of the optical trap in the complex cellular environment, taking into account any variations among cargoes and local viscoelastic properties of the cytoplasm. We found that centrally and peripherally directed forces exerted on LBCs are of similar magnitude, with maximum forces of ∼20 pN. During force events greater than 10 pN, we often observe 8-nm steps in both directions, indicating that the stepping of multiple motors is correlated. These observations suggest bidirectional transport of LBCs is driven by opposing teams of stably bound motors that operate near force balance.

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

“…While this paper was in submission, a similar paper using a similar technique was published (31). The other paper's technique involves measuring the passive and forced response of a trapped cargo in vivo, over many frequencies, and then applying a global fit over the frequencies measured.…”

Identification of a Novel Site in the Tail of Dynein Heavy Chain Important for Dynein Function in Vivo

“…Thus, our current result provides a strong support for the idea that besides its binding to other subunits, the tail is important for regulating dynein motor function in vivo. It is worth pointing out that in vivo, the dynein motor carrying a cargo not only needs to move in a cytoplasm with considerable viscosity (63,64) but also needs to compete with plus-end-directed motors bound to the same cargo (65)(66)(67). In filamentous fungi, kinesin-3 is able to bind to the dynein-bound early endosome and be carried to the minus end as a cargo of dynein (40,53,68).…”

Myosin-I molecular motors at a glance

“…In other words, Myo1b is termed a 'tension-sensitive tether' because when experiencing no mechanical force, the motor detaches from actin filaments within a second after binding to actin and undergoing its powerstroke; however, under resistive loads as low as 1 pN, Myo1b remains anchored to actin filaments for nearly 100 s. These low forces are well within the range of those expected to take place during intracellular transport and mechanotransduction (Gillespie and Cyr, 2004;Hendricks et al, 2012;Soppina et al, 2009). Recent structural and biophysical studies have shown that the tension sensitivity of Myo1b is due in part to a structural element located within the N-terminus of the motor Shuman et al, 2014).…”