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.

Figure 1.

Different force mechanisms that result in MT buckling. (A) A MT can polymerize against a stationary distal tip and buckle as the MT grows. The rate of polymerization slows down as the MT buckles. (B) Acto-myosin contractility can cause a cross-linked MT to buckle. An actin-based motor, such as myosin II, can cross-link antiparallel actin filaments and contract them as it moves. As a result, a MT that is passively attached to the actin will buckle as shown in the figure. (C1a) An MT-based motor such as dynein can walk toward the minus end of a MT, which is cross-linked at the plus end, and cause it to bend. (C1b) The plus-end–directed motion of an actin based motor, such as myosin V, can cause a MT that is cross-linked to the actin to buckle. (C2a) A plus-end–directed MT-based motor, such as kinesin, can buckle a MT that is cross-linked at its minus end to the actin. (C2b) The plus-end–directed motion of an actin based motor also can buckle a MT that is oriented antiparallel to the actin filament and cross-linked at its minus end.

Figure 2.

Direct observation of MT bending in LLC-PK1 cells. (A) A typical digital image of a GFP-tubulin labeled LLC-PK1 epithelial cell showing a broad distribution of MT bending in the periphery of a lamellar extension. To remain in focus, the z-coordinates must remain within ∼0.5 μm of each other, and so the microtubules that are in focus all along their length can be approximated as deforming in the x-y plane only. (B) A closer view of the MT outlined by the box in A. The x-y coordinates determined using the semiautomated algorithm are shown (in red) over the microtubule. The curvature of a MT is estimated locally by collecting a discrete set of x-y coordinates spaced 16 pixels apart along the contour (blue dots) and calculating the angle change with respect to the average arc length of three adjacent points (see equation 2). Often, highly bent MTs (MT 1) are observed directly adjacent to straight MTs (MT 2). Microtubules were not selected for analysis based on their curvature. (C) A noncentrosomal MT increases its bending during depolymerization, indicating that polymerization-based forces are unlikely to drive bending in all cases. (D) A highly bent MT rapidly relaxes after the tip depolymerizes past a putative attachment point, providing evidence that MTs are both stiff and cross-linked to other structures in the cell. Bar in all images, 3 μm.

Figure 3.

F-actin and microtubule dynamics in LLC-PK1 cells. (A) The top two images from the montage show the first frame in a time series of actin (left) and tubulin (right) channels in a typical cell dually labeled with mCherry-actin and EGFP-tubulin. The middle frames show both actin and microtubule dynamics, respectively, at frame 50. The bottom frames show 50 consecutive frames of actin and microtubule dynamics, respectively, compressed into one image via a max projection (1 min 38 s elapsed time). Note that specific features (shown with red arrows) in the actin channel seem very similar in the before and after images, suggesting that actin does not move significantly over the time of the movie. In addition, the max projection image shows virtually no blurring of the actin channel due to F-actin movement. This stands in contrast to the tubulin channel before and after images where significant redistribution of the MT array results in completely different images. In addition, the max projection in the tubulin channel shows marked blurring of individual microtubules arising from movement of individual MTs during the course of the movie. (B) Typical image from an mCherry-actin–labeled LLC-PK1 cell. Time-lapse images were taken every 400 ms, and a kymograph (C) of F-actin motion was made. The white rectangle in B shows the region from which the kymograph in C was created. The F-actin kymograph shows that the actin retrograde flow is very small (∼7 nm/s), which is too slow to drive MT bending. Horizontal bar, 5 μm (A) and 3 μm (B and C). Vertical bar, 10 s (C).

Figure 4.

Microtubule deformation in the absence of myosin II activity. (A) The image from a GFP-tubulin–labeled LLC-PK1 epithelial cell showing the distribution and the bending of microtubules near the cell periphery before the drug treatment. (B) The image of the same cell 15 min after the addition of blebbistatin. Time (minutes) is in relation to the addition of blebbistatin at a concentration of 75 μM. Horizontal bar, 5 μm.

Figure 5.

MT bending dynamics in LLC-PK1 Cells. (A) Montage of a speckled MT bending and unbending. The white box represents the region from which the kymograph is created. Note that the white box is sufficiently wide so that it encompasses the entire bending microtubule for the duration of the movie. Bar, 3 μm. (B) Kymograph of the speckled microtubule in A showing that anterograde transport of the proximal microtubule coupled with a stationary distal plus end drives MT bending. Magenta arrow indicates t = 18 s from A.

Figure 6.

Microtubule deformation in the absence of polymerization. Distribution of microtubules in the periphery of GFP-tubulin–labeled LLC-PK1 epithelial cells is shown before and after exposure to different concentrations of the drug nocodazole. (A) Nocodazole is used at 50 μM. (B) Nocodazole is used at 100 nM. Time (minutes) is in relation to the addition of nocodazole. Horizontal bar, 5 μm.

Figure 7.

Bending dynamics of nonpolymerizing MTs in LLC-PK1 cells. Nocodazole at a concentration of 100 nM is used to suppress dynamic instability. (A) Montage of a speckled MT being transported anterogradely. The white box represents the region from which the kymograph is created. The blue and red boxes denote frames that correspond to t = 12 and t = 60 s, respectively. Bar, 3 μm. (B) Kymograph of the microtubule in A showing the anterograde transport of the proximal microtubule at two different times, t = 12 s (shown by the blue arrow) and t = 60 s (shown by the red arrow), against a stationary distal plus end (yellow line).

Figure 8.

Microtubule bending mechanisms and transport direction. (A) Histogram of microtubule bending mechanisms from kymographic analysis of deforming microtubules (n = 33 MTs). Blue shows the number of MTs in which the proximal portion of the microtubule is being transported anterogradely against a stationary distal tip, whereas red shows the number of MTs in which the distal portion is being transported retrogradely against a stationary proximal portion, and magenta shows the number of both mechanisms working together to drive microtubule bending. (B) Histogram showing microtubule transport regardless of bend formation (n = 77 MTs). Green shows the number of microtubules that do not move over the observation period; blue and red show the number of microtubules that move in an anterograde and retrograde direction, respectively; and maroon shows the number of microtubules that move bidirectionally.

Figure 9.

MTs in gliding assays in vitro. (A) Typical digital image of a MT-kinesin gliding assay. Some MTs are highly bent, whereas other MTs are relatively straight, and MTs qualitatively seem to be deformed in a manner similar to that shown in vivo (see Figure 2A). (B) MT gliding in the direction of the arrow is cross-linked, presumably by a dead motor, at its leading tip for the first three frames while curvature develops in the MT. In the fourth and subsequent frames, the cross-link is lost, and the MT springs forward. Arrowheads denote the position of the putative cross-link. (C) A bent MT gliding in the direction of the arrow, glides past a cross-link site (arrowhead), and rapidly relaxes similar to the relaxing MT in Figure 2D. Bar, 3 μm.

Figure 10.

MT curvature distribution in living cells and gliding assays. MT bending in LLC-PK1 cells was quantified and the curvature distribution was constructed using n = 125 individual MTs from n = 6 cells. A spacing of 16 pixels corresponding to 0.67 μm was used. The curvature distributions in vivo and in vitro are shown in black and red, respectively. The experimental curvature distribution was compared with a thermal model (Gaussian) of MT bending using the flexural rigidity of Taxol-stabilized MTs (EI ∼2 pN-m²) and showed poor agreement (shown in dashed lines), especially in the tail of the distribution.

Figure 11.

Cartoon representation of microtubule bending and transport in an LLC-PK1 cell. The motor shown in (blue) is a minus-end–directed motor, such as dynein. A typical MT cross-linked at the distal tip, buckles as a result of the motor force. The MT based motors anchored at the actin rich cortex cause the deformation of the MTs in the middle regions of the cell. The force on the given MT is presumably balanced by cross-links on MTs on the other side of the cell. Some of these forces can also presumably be accommodated by the centrosome or linkages to it.