In this study, we characterized the dynamics and organization of F-actin, myosin, and α-actinin in the formation of contractile lamellar networks and stress fibers that generate and sustain varying levels of cellular tension. Altogether we describe two distinct phases of tension buildup: the first, at low tension, is governed primarily by changes in lamellar actomyosin dynamics; the second, at higher tension, is governed primarily by the remodeling of lamellar networks into stress fibers.
At the lowest force levels, the lamellar actomyosin network underwent rapid flow, at maximum rates of ~70 nm/s, consistent with the unloaded speed of nonmuscle myosin II measured in vitro (Cuda et al.
). This retrograde flow decayed fourfold as cellular force generation increased. During this time, thin bundles formed in the lamellar network and focal adhesions elongated. The changes in dynamics suggest that, during this stage, myosin motors rapidly contract an actin meshwork until they stall. An estimation of the stall force of myosin motors in the cell is approximated by Fcell
, where Fs
is the stall force of the motor, A is the cell area, npuncta
is the density of myosin puncta in the lamella, and Nmyosin
is the number of myosin motors per puncta. Indeed using experimentally measured values of A ~ 3600 μm2
~ 1 puncta/μm2
along with reported estimates of Fs
~ 2 pN (Molloy et al.
) and Nmyosin
~ 20 (Niederman and Pollard, 1975
; Verkhovsky and Borisy, 1993
), the number we obtain is on the order of 80–100 nN, consistent with the total cellular traction exerted at this time. We speculate that this stall force sets the transition when retrograde flow is stabilized and remodeling of the network into bundles commences. Thus over half of the total force output of the cell can be obtained through modifications to retrograde flow dynamics of a contractile lamellar actomyosin network ().
FIGURE 7: Correlation between cellular tension states and changes in cytoskeletal organization and dynamics. At low tension and rapid time scales, increased cellular force generation is inversely correlated to retrograde flow dynamics in a contractile actin network. (more ...)
When the retrograde flow of actin and myosin flow stabilizes, lamellar actin is remodeled into a network of stress fibers spanning the lamella and cell body (). This remodeling requires sufficiently stiff ECM and is correlated to moderate changes in cellular tension over long time scales. During this period, both the number of actin bundles increases and individual bundles thicken. During bundle thickening, bundles accumulate actin, myosin band spacing decreases, and α-actinin bands form and intensify. At the highest levels of cellular tension, a network of stress fibers with alternating bands of α-actinin and myosin with a spacing of ~1 μm is formed.
Our data strongly suggest a model of stress fiber formation whereby myosin remodels actin into thin bundles, which then promotes the accumulation of α-actinin. We speculate that myosin-mediated alignment of F-actin into compact parallel and/or antiparallel filaments facilitates the enhanced binding of α-actinin, which favors the binding of closely spaced F-actin (Bartles, 2000
). Future work is required to test this speculation and to rule out other mechanisms of α-actinin recruitment. Our model contrasts previous models, which suggest that α-actinin bundling of F-actin precedes the incorporation of myosin (Hotulainen and Lappalainen, 2006
). One possibility for this difference is that previous experiments studied stress fiber formation near the leading cell edge. Near the leading edge, stress fiber assembly occurs rapidly within a small region. Thus low-tension actomyosin dynamics and organization could be difficult to assess.
We speculate that the mechanisms for remodeling of the lamellar meshwork into actin bundles is likely to occur both by the realignment of preexisting F-actin into bundles by myosin (Verkhovsky et al.
) and by the polymerization dynamics of F-actin (Hotulainen and Lappalainen, 2006
). Although it is difficult to directly assess the former, photobleaching measurements have shown that actin in lamellar contractile networks turns over quite rapidly (~30 s), making it unlikely that individual actin filaments are stable during the 10 min of bundle assembly and that mechanisms to promote actin assembly along the entire bundle length during thickening are necessary. Dissecting the molecular mechanisms that control this accretion process requires future work.
A surprising result stemming from our study is that a significant amount of force can be generated in a contractile lamellar network absent of stress fibers. Moreover, this force generation appears to be unaffected by changes in ECM compliance. This finding suggests that one must reconsider the assumptions of a cellular contractile state based solely on the prevalence of stress fibers. We speculate that the contractile lamellar networks may allow for rapid tuning of cytoskeletal tension in response to changes in external or internal forces over second time scales. By contrast, the slow dynamics of stress fiber assembly may not facilitate such rapid tuning.
Our results reveal a continuum of actomyosin organizations at different levels of tension with contractile lamellar networks and networks of stress fibers characterizing the lower and upper bounds, respectively. These bounds can be described by common cell types, such as keratocytes or Ptk1 cells, which exert low levels of force and tend to form contractile lamellar networks (Theriot and Mitchison, 1991
; Lee et al.
), and fibroblasts, which can exert high levels of force and typically display abundant stress fibers (Pelham and Wang, 1997
; Balaban et al.
; Tan et al.
). Consistent with our data, traction forces in keratocytes and Ptk1 cells are largely regulated by actin dynamics (Gardel et al.
; Fournier et al.
), consistent with several recent models of actomyosin contractility (Kruse et al.
; Rubinstein et al.
). By contrast, we expect forces in stress fiber–rich cells to be regulated by structural effects, as speculated by other models of cellular force generation (Bischofs et al.
). Our results indicate that both types of models are needed to capture the biophysical behavior over a large range of forces and, ideally, these would be integrated to take into account large rearrangements in forces observed during dynamic cellular processes. From this, a coherent biophysical understanding of force generation by the actomyosin cytoskeleton will likely emerge.