Microtubule guidance tested through controlled cell geometry

Abstract

SummaryIn moving cells dynamic microtubules (MTs) target and disassemble substrate adhesion sites (focal adhesions; FAs) in a process that enables the cell to detach from the substrate and propel itself forward. The short-range interactions between FAs and MT plus ends have been observed in several experimental systems, but the spatial overlap of these structures within the cell has precluded analysis of the putative long-range mechanisms by which MTs growing through the cell body reach FAs in the periphery of the cell. In the work described here cell geometry was controlled to remove the spatial overlap of cellular structures thus allowing for unambiguous observation of MT guidance. Specifically, micropatterning of living cells was combined with high-resolution in-cell imaging and gene product depletion by means of RNA interference to study the long-range MT guidance in quantitative detail. Cells were confined on adhesive triangular microislands that determined cell shape and ensured that FAs localized exclusively at the vertices of the triangular cells. It is shown that initial MT nucleation at the centrosome is random in direction, while the alignment of MT trajectories with the targets (i.e. FAs at vertices) increases with an increasing distance from the centrosome, indicating that MT growth is a non-random, guided process. The guided MT growth is dependent on the presence of FAs at the vertices. The depletion of either myosin IIA or myosin IIB results in depletion of F-actin bundles and spatially unguided MT growth. Taken together our findings provide quantitative evidence of a role for long-range MT guidance in MT targeting of FAs.

Adhesive interactions of N-cadherin limit the recruitment of microtubules to cell–cell contacts through organization of actomyosin

“…Indeed, MTs have been reported to be targeted to and captured at cellextracellular-matrix focal adhesions (Kaverina et al, 1998). MTs have been implicated in signaling, relaxation and trafficking at integrin-mediated adhesions, leading to destabilization of focal adhesions and an increased rate of turnover, a process that is essential for cell polarization and migration (Huda et al, 2012;Kaverina et al, 1998;Small and Kaverina, 2003;Stehbens and Wittmann, 2012). However, the relationship between cadherins and MTs is still unclear and controversial.…”

Adhesive interactions of N-cadherin limit the recruitment of microtubules to cell–cell contacts through organization of actomyosin

“…The major obstacle to MT penetration into areas containing cadherin adhesions was not the reduction in the stability of plus ends and polymerization, but, instead, seemed to be the physical barrier formed by circular actin cables at the rear of the adhesion area. Indeed, in cells seeded on FN, it has been shown that MTs are guided along stress fibers to reach the leading edge (Huda et al, 2012). By contrast, cells on Ncad-Fc do not form longitudinal stress fibers but, instead, have actin filaments that are organized in a circular dense actomyosin belt.…”

Microfabricated Systems and Assays for Studying the Cytoskeletal Organization, Micromechanics, and Motility Patterns of Cancerous Cells

“…[51–53] In the context of cell motility, recent studies have shown that MTs not only play a role in the formation of lamellipodial protrusions at the front of a moving cell, [58] but are also important in the disassembly of focal adhesions (FAs) at the cell’s rear. [51,59,60] …”

“…In contrast, the rear of the cell is characterized by frequent MT catastrophes, induced by excess of active catastrophe-inducing factors, such as stathmin [61] and also by contact with focal adhesions. [62] MTs located there in are guided towards, [60] target focal adhesions [63] (with which cell attaches to the substrate, see Figure 5 and discussion in the next paragraph) and help to disassemble these multimolecular complexes [51] allowing the cell to retract its tail and ultimately move forward. For excellent discussion of asymmetric microtubule dynamics in migrating cells, the Reader is directed to review [74] .…”

“…For excellent discussion of asymmetric microtubule dynamics in migrating cells, the Reader is directed to review [74] . In Section 6.1 of this review, we describe an approach used to quantify MT guidance towards FAs by tracking MT trajectories in micropatterned triangular cells [60] in Section 6.1. Other interesting systems, designed to study how MTs interact with cell boundary, include microfabricated chambers in which in vitro polymerized MTs interact with and exert forces against functionalized barriers that mimic cell boundary.…”