Microtubules (MTs) are ubiquitous cytoskeletal polymers in eukaryotic cells that consist of α/β tubulin heterodimers assembled head-to-tail in the 13 protofilaments making up the 25-nm-radius cylindrical MT wall. Both α- and β-tubulin bind GTP, and the relationship between tubulin GTP hydrolysis, MT assembly, and MT stability results in a behavior known as “dynamic instability,” in which growing and shrinking MTs coexist in a population when MTs are in equilibrium with tubulin dimer. In such a population, individual MTs constantly make stochastic transitions between persistent phases of growth and shortening (reviewed in Desai and Mitchison, 1997
). The kinetic parameters describing dynamic instability include the velocities of MT growth and shortening and the frequencies of transition between growth and shortening (catastrophe frequency) and between shortening and growth (rescue frequency) (Walker et al., 1988
). In addition, the intrinsic polarity of tubulin heterodimers and their unidirectional orientation during association results in a MT polymer with structural polarity, such that dimers add more quickly to the “plus” end and more slowly to the “minus” end (Walker et al., 1988
). In vivo, this MT polarity is thought to lend overall polarity and organization to living cells. For example, in tissue cells in culture, MTs are organized with their minus ends either bound to the centrosome adjacent to the nucleus or free and facing toward the cell center and their plus ends radiating out toward the cell periphery (Euteneuer and McIntosh, 1981
Major questions in MT cell biology include the physiological role of MT plus end dynamic instability and its regulation. Cycles of growth and shortening of MT plus ends during dynamic instability may be a means for MTs to “search” cellular space, as in the case of chromosome kinetochore capture by dynamically unstable MTs during the establishment of the mitotic spindle (reviewed in Rieder and Salmon, 1998
) or in the targeting of MTs to focal contacts and promotion of focal contact disassembly in migrating cells (Kaverina et al., 1998
). Alternatively, recent evidence suggests that certain proteins can bind to MT ends only during specific phases of dynamic instability (Perez et al., 1999
). Further, MT plus end growth and shortening may activate different signal transduction cascades to produce differential regulation of the actin cytoskeleton (Ren et al., 1999
; Waterman-Storer et al., 1999
; reviewed in Waterman-Storer and Salmon, 1999
). In either case, the glorious advantage of the dynamically unstable MT plus end is its exquisite spatial resolution, making MT dynamic instability a prime candidate for precise control of spatial regulatory processes in the cell. For example, in polarized migrating cells with a free leading edge exhibiting actomyosin-ruffling activity, MTs align along the axis of migration, with their plus ends facing the direction of cell movement (Gotlieb et al., 1981
; Kupfer et al., 1982
). These polar MTs could then precisely direct the delivery of signaling molecules to drive ruffling, structural components of the motile machinery, or regulators of focal contacts that are required at specific sites at or near the leading edge.
In addition, it has been proposed that selective stabilization of the dynamic instability of individual MT plus ends in specific regions of the cell may promote the establishment of such cellular asymmetries (Kirschner and Mitchison, 1986
). Thus, in order to understand the spatial organization of cells, it is of prime importance to understand the regulation of MT plus end dynamic instability in vivo. It is well established that the parameters of MT dynamic instability differ for pure tubulin in vitro and MTs in living cells. Indeed, several protein factors that bind to either tubulin dimers or MT polymer (MAPs) have been identified that regulate specific phases of dynamic instability, such as promotion of catastrophe, enhancing the rates of growth/shortening, or suppressing rescue (reviewed in Cassimeris, 1999
). However, the cellular events and/or cellular contexts that regulate these proteins are unclear.
One possible cellular context that may modulate plus end MT dynamic instability is whether cells exist as part of a tissue or are free in culture. To approach this question, we were interested to know whether the dynamic instability of individual MT plus ends was altered by cell–cell contact. In tissues and in culture, contacts between cells are mediated by morphologically distinct structures, including tight junctions, adherens junctions, and desmosomes. They all consist, in some manner, of trans-membrane receptors mediating cell–cell interaction on the outside of the cell, whereas on the inside of the cell, they mediate connections to the cortical cytoskeleton. Tight junctions (TJs) form around the apical domain between polarized epithelial cells and seal the cells' apical surface from their basolateral side. TJs are made up of trans-membrane proteins occludin and claudin, which bind in the cytoplasm to membrane-associated guanylate cyclase kinase homologues, including ZO-1 and ZO-2, which may link to cortical actin filaments (reviewed in Tsukita and Furuse, 1999
). Adherens junctions, which do not form a seal, but only anchor neighboring cells to one another, consist of trans-membrane cadherins (E-, N-, and VE-cadherin) that bind to intracellular catenins (α-catenin, β-catenin, plakoglobin), which link to cortical actin via either direct (through β-catenin) or indirect (through vinculin or α-actinin) interactions (Provost and Rimm, 1999
; reviewed in Steinberg and McNutt, 1999
). Desmosomes consist of trans-membrane desmogleins that interact with desmoplakins, which link to intermediate filaments (reviewed in Troyanovsky, 1999
In the present study, we were interested to know if the dynamic instability of MT plus ends in cells in the center of the sheet that were contacted on all sides by neighboring cells differed from the dynamic behavior of MT plus ends at the leading free edge of a sheet of squamous epithelial cells migrating during a wound healing reaction in culture. Our results show that plus end dynamic instability is suppressed in fully contacted cells, with individual MTs exhibiting an extended state of pause, suggesting that they become capped. We also find that depolymerization of MTs in fully contacted cells induces disruption of cell–cell adherens junctions. This suggests that a feedback relationship exists in which MT dynamics are modulated by cell–cell contact, and the maintenance of contacts require MTs.