Anterograde Microtubule Transport Drives Microtubule Bending in LLC-PK1 Epithelial Cells

Abstract

Microtubules (MTs) have been proposed to act mechanically as compressive struts that resist both actomyosin contractile forces and their own polymerization forces to mechanically stabilize cell shape. To identify the origin of MT bending, we directly observed MT bending and F-actin transport dynamics in the periphery of LLC-PK1 epithelial cells. We found that F-actin is nearly stationary in these cells even as MTs are deformed, demonstrating that MT bending is not driven by actomyosin contractility. Furthermore, the inhibition of myosin II activity through the use of blebbistatin results in microtubules that are still dynamically bending. In addition, as determined by fluorescent speckle microscopy, MT polymerization rarely results, if ever, in bending. We suppressed dynamic instability using nocodazole, and we observed no qualitative change in the MT bending dynamics. Bending most often results from anterograde transport of proximal portions of the MT toward a nearly stationary distal tip. Interestingly, we found that in an in vitro kinesin-MT gliding assay, MTs buckle in a similar manner. To make quantitative comparisons, we measured curvature distributions of observed MTs and found that the in vivo and in vitro curvature distributions agree quantitatively. In addition, the measured MT curvature distribution is not Gaussian, as expected for a thermally driven semiflexible polymer, indicating that thermal forces play a minor role in MT bending. We conclude that many of the known mechanisms of MT deformation, such as polymerization and acto-myosin contractility, play an inconsequential role in mediating MT bending in LLC-PK1 cells and that MT-based molecular motors likely generate most of the strain energy stored in the MT lattice. The results argue against models in which MTs play a major mechanical role in LLC-PK1 cells and instead favor a model in which mechanical forces control the spatial distribution of the MT array.

Loop formation of microtubules during gliding at high density

“…This is reminiscent of the curvatures that occur in gliding assays when microtubules are pinned by a dead motor. This analogy between gliding assays and in vivo microtubule bending has recently been explored using curvature distributions of microtubules located in the periphery of LLC-PK1 epithelial cells (Bicek et al 2007(Bicek et al , 2009) and in kinesin gliding assays. It was shown that microtubule curvature distributions in vivo and in vitro both exhibited an exponential decay, indicating the involvement of active forces.…”

Endocytic membrane fusion and buckling-induced microtubule severing mediate cell abscission

“…Despite the fact that the cause of microtubule buckling remains elusive, previous studies suggested mechanisms mediating microtubule bending and buckling. It has been shown that kinesin and dynein motors might result in microtubule buckling and breaking during telophase (Bicek et al, 2009). Because several microtubule motors are known to be necessary for the completion of cytokinesis (Zhu et al, 2005), perhaps these microtubule motors might also contribute to the central spindle buckling by either anchoring themselves to the PM or by anchoring to another microtubule to create a bending force.…”

Functional coupling of microtubules to membranes – implications for membrane structure and dynamics

“…Microtubule sliding is central to the organization of the mitotic spindle and, here, might have a role in organelle partitioning during mitosis (see Goshima et al, 2005). The impact of motor force on microtubule structure would also have indirect consequences for any attached organelle (Bicek et al, 2009). A further example is provided by the attachment of the ER to microtubule tips, which results in the extension of the ER in response to microtubule polymerization (Grigoriev et al, 2008).…”