Mechanoaccumulative Elements of the Mammalian Actin Cytoskeleton

Abstract

To change shape, divide, form junctions, and migrate, cells reorganize their cytoskeletons in response to changing mechanical environments [1-4]. Actin cytoskeletal elements, including myosin II motors and actin crosslinkers, structurally remodel and activate signaling pathways in response to imposed stresses [5-9]. Recent studies demonstrate the importance of force-dependent structural rearrangement of α-catenin in adherens junctions [10] and vinculin's molecular clutch mechanism in focal adhesions [11]. However, the complete landscape of cytoskeletal mechanoresponsive proteins and the mechanisms by which these elements sense and respond to force remain to be elucidated. To find mechanosensitive elements in mammalian cells, we examined protein relocalization in response to controlled external stresses applied to individual cells. Here, we show that non-muscle myosin II, α-actinin, and filamin accumulate to mechanically stressed regions in cells from diverse lineages. Using reaction-diffusion models for force-sensitive binding, we successfully predicted which mammalian α-actinin and filamin paralogs would be mechanoaccumulative. Furthermore, a Goldilocks zone must exist for each protein where the actin-binding affinity must be optimal for accumulation. In addition, we leveraged genetic mutants to gain a molecular understanding of the mechanisms of α-actinin and filamin catch-bonding behavior. Two distinct modes of mechanoaccumulation can be observed: a fast, diffusion-based accumulation and a slower, myosin II-dependent cortical flow phase that acts on proteins with specific binding lifetimes. Finally, we uncovered cell-type and cell-cycle-stage-specific control of the mechanosensation of myosin IIB, but not myosin IIA or IIC. Overall, these mechanoaccumulative mechanisms drive the cell's response to physical perturbation during proper tissue development and disease.

The fifth sense: Mechanosensory regulation of alpha-actinin-4 and its relevance for cancer metastasis

“…Beyond this threshold, the bond ruptures, decreasing the bond lifetime. In fact, the loss of the salt bridge in the ACTN4 K255E mutant disrupts α-actinin- 4’s mechanoresponsiveness, further supporting this molecular explanation for the catch-slip bond function necessary for mechanosensitive accumulation [27]. The mechanosensitive accumulation appears to depend on catch-slip bond formation as well as an optimal actin-binding affinity range, which is observed for wild type ACTN4 (K D = 32 µM) [45] and not ACTN1 (K D = 0.36 µM) [46] (Fig 3).…”

“…What is becoming evident is that targeting one aspect of tumor biology, such as cell division, may be insufficient and both tumor cell and stroma need to be addressed for optimal survivability [25]. ACTN4 is a key link between the two tumor components, because it directs the coupling of the actin cytoskeleton inside the cell to the integrins that directly bind the stromal ECM [26], and is capable of sensing and responding to externally-applied force [27]. For this reason, its mechanisms of regulation may provide insight into methods of uncoupling the two tumor components, and thus inhibiting invasion.…”

“…Historically, four separate mechanisms have been described: phosphorylation of specific residues, calcium modulation, cleavage by calpain, and binding of lipids such as PIP3. Recent evidence suggests that mechanical stress results in a fifth mode of α-actinin regulation [11, 27]. The following sections will describe how each individual mode of regulation affects actin binding and discuss how these mechanisms are combined to fine tune the activity of ACTN4.…”

“…Disruption of this salt bridge exposes the second actin-binding sequence, allowing it to bind, and thereby increasing α-actinin-4’s actin affinity [45]. This shift in binding affinity upon conformational change could be the molecular mechanism underlying α-actinin’s mechanoresponse, defined as the ability of α-actinin to accumulate in response to applied mechanical stress [11, 27]. The mechanoresponsiveness implicates catch-slip bond behavior between α-actinin and actin [11].…”

“…The mechanosensitive accumulation appears to depend on catch-slip bond formation as well as an optimal actin-binding affinity range, which is observed for wild type ACTN4 (K D = 32 µM) [45] and not ACTN1 (K D = 0.36 µM) [46] (Fig 3). Without the catch-slip bond, no diffusion-mediated mechanoaccumulation is observed, as is the case with the ACTN4 K255E mutant which has a higher affinity to actin (K D = 7 µM) [45], but predicted to have no catch bond activity [27]. On the other hand, if the actin-binding affinity is too high, then mechanoresponsiveness is abolished by the absence of an available free pool of α-actinin, as seen with the high affinity paralog, ACTN1.…”