Elizabeth Blankman

A Zyxin-Mediated Mechanism for Actin Stress Fiber Maintenance and Repair

To maintain mechanical homeostasis, cells must recognize and respond to changes in cytoskeletal integrity. By imaging live cells expressing fluorescently tagged cytoskeletal proteins, we observed that actin stress fibers undergo local, acute, force-induced elongation and thinning events that compromise their stress transmission function, followed by stress fiber repair that restores this capability. The LIM protein zyxin rapidly accumulates at sites of strain-induced stress fiber damage and is essential for stress fiber repair and generation of traction force. Zyxin promotes recruitment of the actin regulatory proteins α-actinin and VASP to compromised stress fiber zones. α-Actinin plays a critical role in restoration of actin integrity at sites of local stress fiber damage, whereas both α-actinin and VASP independently contribute to limiting stress fiber elongation at strain sites, thus promoting stabilization of the stress fiber. Our findings demonstrate a mechanism for rapid repair and maintenance of the structural integrity of the actin cytoskeleton.

Zinc fingers can act as Zn2+ sensors to regulate transcriptional activation domain function

The yeast Zap1 transcription factor controls the expression of genes involved in zinc accumulation and storage. Zap1 is active in zinc‐limited cells and repressed in replete cells. Zap1 has two activation domains, AD1 and AD2, which are both regulated by zinc. AD2 function was mapped to a region containing two Cys2His2 zinc fingers, ZF1 and ZF2, that are not involved in DNA binding. More detailed mapping placed AD2 almost precisely within the endpoints of ZF2, suggesting a role for these fingers in regulating activation domain function. Consistent with this hypothesis, ZF1 and ZF2 bound zinc in vitro but less stably than did zinc fingers involved in DNA binding. Furthermore, mutations predicted to disrupt zinc binding to ZF1 and/or ZF2 rendered AD2 constitutively active. Our results also indicate that the repressed form of AD2 requires an intramolecular interaction between ZF1 and ZF2. These studies suggest that these zinc fingers play an unprecedented role as zinc sensors to control activation domain function.

The Zap1 transcriptional activator also acts as a repressor by binding downstream of the TATA box in ZRT2

The zinc‐responsive transcriptional activator Zap1 regulates the expression of both high‐ and low‐affinity zinc uptake permeases encoded by the ZRT1 and ZRT2 genes. Zap1 mediates this response by binding to zinc‐responsive elements (ZREs) located within the promoter regions of each gene. ZRT2 has a remarkably different expression profile in response to zinc compared to ZRT1. While ZRT1 is maximally induced during zinc limitation, ZRT2 is repressed in low zinc but remains induced upon zinc supplementation. In this study, we determined the mechanism underlying this paradoxical Zap1‐dependent regulation of ZRT2. We demonstrate that a nonconsensus ZRE (ZRT2 ZRE3), which overlaps with one of the ZRT2 transcriptional start sites, is essential for repression of ZRT2 in low zinc and that Zap1 binds to ZRT2 ZRE3 with a low affinity. The low‐affinity ZRE is also essential for the ZRT2 expression profile. These results indicate that the unusual pattern of ZRT2 regulation among Zap1 target genes involves the antagonistic effect of Zap1 binding to a low‐affinity ZRE repressor site and high‐affinity ZREs required for activation.

Mapping the DNA Binding Domain of the Zap1 Zinc-responsive Transcriptional Activator

The Zap1 transcriptional activator ofSaccharomyces cerevisiae plays a major role in zinc homeostasis by inducing the expression of several genes under zinc-limited growth conditions. This activation of gene expression is mediated by binding of the protein to one or more zinc-responsive elements present in the promoters of its target genes. To better understand how Zap1 functions, we mapped its DNA binding domain using a combined in vivo and in vitro approach. Our results show that the Zap1 DNA binding domain maps to the carboxyl-terminal 194 amino acids of the protein; this region contains five of its seven potential zinc finger domains. Fusing this region to the Gal4 activation domain complemented a zap1Δ mutation for low zinc growth and also conferred high level expression on a zinc-responsive element-lacZ reporter. In vitro, the purified 194-residue fragment bound to DNA with a high affinity (dissociation constant in the low nanomolar range) similar to that of longer fragments of Zap1. Furthermore, by deletion and site-directed mutagenesis, we demonstrated that each of the five carboxyl-terminal zinc fingers are required for high affinity DNA binding.

A mechanical-biochemical feedback loop regulates remodeling in the actin cytoskeleton

Significance Cellular activities are regulated by signaling pathways in which information is transduced biochemically. Increasingly it is appreciated that regulation also involves mechanical signaling, where mechanical information is mechanotransduced into biochemical information. However, little is understood about the cooperation of mechanical and biochemical signaling in mixed pathways. We identified a pathway where the two types of signaling work in harmony to remodel actomyosin stress fibers in the cell’s cytoskeleton. We present evidence that expansion or contraction of fibers alters the actin filament overlap, a mechanical signal that is mechanotransduced into actin assembly or disassembly, which in turn alters the overlap. A mathematical model accurately describes our measurements and shows that this mechanical-biochemical feedback loop synchronizes actin remodeling with fiber length changes. Cytoskeletal actin assemblies transmit mechanical stresses that molecular sensors transduce into biochemical signals to trigger cytoskeletal remodeling and other downstream events. How mechanical and biochemical signaling cooperate to orchestrate complex remodeling tasks has not been elucidated. Here, we studied remodeling of contractile actomyosin stress fibers. When fibers spontaneously fractured, they recoiled and disassembled actin synchronously. The disassembly rate was accelerated more than twofold above the resting value, but only when contraction increased the actin density to a threshold value following a time delay. A mathematical model explained this as originating in the increased overlap of actin filaments produced by myosin II-driven contraction. Above a threshold overlap, this mechanical signal is transduced into accelerated disassembly by a mechanism that may sense overlap directly or through associated elastic stresses. This biochemical response lowers the actin density, overlap, and stresses. The model showed that this feedback mechanism, together with rapid stress transmission along the actin bundle, spatiotemporally synchronizes actin disassembly and fiber contraction. Similar actin remodeling kinetics occurred in expanding or contracting intact stress fibers but over much longer timescales. The model accurately described these kinetics, with an almost identical value of the threshold overlap that accelerates disassembly. Finally, we measured resting stress fibers, for which the model predicts constant actin overlap that balances disassembly and assembly. The overlap was indeed regulated, with a value close to that predicted. Our results suggest that coordinated mechanical and biochemical signaling enables extended actomyosin assemblies to adapt dynamically to the mechanical stresses they convey and direct their own remodeling.

LIM Domains Target Actin Regulators Paxillin and Zyxin to Sites of Stress Fiber Strain

Contractile actomyosin stress fibers are critical for maintaining the force balance between the interior of the cell and its environment. Consequently, the actin cytoskeleton undergoes dynamic mechanical loading. This results in spontaneous, stochastic, highly localized strain events, characterized by thinning and elongation within a discrete region of stress fiber. Previous work showed the LIM-domain adaptor protein, zyxin, is essential for repair and stabilization of these sites. Using live imaging, we show paxillin, another LIM-domain adaptor protein, is also recruited to stress fiber strain sites. Paxillin recruitment to stress fiber strain sites precedes zyxin recruitment. Zyxin and paxillin are each recruited independently of the other. In cells lacking paxillin, actin recovery is abrogated, resulting in slowed actin recovery and increased incidence of catastrophic stress fiber breaks. For both paxillin and zyxin, the LIM domains are necessary and sufficient for recruitment. This work provides further evidence of the critical role of LIM-domain proteins in responding to mechanical stress in the actin cytoskeleton.

Zinc-regulated DNA binding of the yeast Zap1 zinc-responsive activator.

The Zap1 transcription factor of Saccharomyces cerevisiae plays a central role in zinc homeostasis by controlling the expression of genes involved in zinc metabolism. Zap1 is active in zinc-limited cells and repressed in replete cells. At the transcriptional level, Zap1 controls its own expression via positive autoregulation. In addition, Zap1s two activation domains are regulated independently of each other by zinc binding directly to those regions and repressing activation function. In this report, we show that Zap1 DNA binding is also inhibited by zinc. DMS footprinting showed that Zap1 target gene promoter occupancy is regulated with or without transcriptional autoregulation. These results were confirmed using chromatin immunoprecipitation. Zinc regulation of DNA binding activity mapped to the DNA binding domain indicating other parts of Zap1 are unnecessary for this control. Overexpression of Zap1 overrode DNA binding regulation and resulted in constitutive promoter occupancy. Under these conditions of constitutive binding, both the zinc dose response of Zap1 activity and cellular zinc accumulation were altered suggesting the importance of DNA binding control to zinc homeostasis. Thus, our results indicated that zinc regulates Zap1 activity post-translationally via three independent mechanisms, all of which contribute to the overall zinc responsiveness of Zap1.

The Cytoskeletal Regulator Zyxin is Required for Viability in Drosophila melanogaster

The zyxin family of proteins function as cytoskeletal regulators in adhesion, actin assembly, and cell motility. Though fibroblasts derived from zyxin‐null mice show striking defects in motility and response to mechanical stimuli, the mice are viable and fertile. In Drosophila melanogaster, the family is represented by a single homologue, Zyx102. To study the role of zyxin during development, we generated a zyx102 RNA‐interference transgenic line that allows for the conditional knockdown of Zyx102. When UAST‐zyx102‐dsRNAi expression is driven broadly by Actin5C‐GAL4, loss of Zyx102 results in lethality during the pharate adult stage, a narrow developmental window during which the fly must molt, resorb molting fluid, fill adult trachea with air, and execute a behavioral program to eclose. Zyx102 knockdown animals attempt to emerge, but their adult trachea do not fill with air. If dissected from the pupal case, knockdown individuals appear morphologically normal, but remain inviable. Anat Rec 293:1455–1469, 2010.

Lateral communication between stress fiber sarcomeres facilitates a local remodeling response.

Actin stress fibers (SFs) are load-bearing and mechanosensitive structures. To our knowledge, the mechanisms that enable SFs to sense and respond to strain have not been fully defined. Acute local strain events can involve a twofold extension of a single SF sarcomere, but how these dramatic local events affect the overall SF architecture is not believed to be understood. Here we have investigated how SF architecture adjusts to episodes of local strain that occur in the cell center. Using fluorescently tagged zyxin to track the borders of sarcomeres, we characterize the dynamics of resting sarcomeres and strain-site sarcomeres. We find that sarcomeres flanking a strain site undergo rapid shortening that directly compensates for the strain-site extension, illustrating lateral communication of mechanical information along the length of a stress fiber. When a strain-site sarcomere extends asymmetrically, its adjacent sarcomeres exhibit a parallel asymmetric shortening response, illustrating that flanking sarcomeres respond to strain magnitude. After extension, strain-site sarcomeres become locations of new sarcomere addition, highlighting mechanical strain as a trigger of sarcomere addition and revealing a, to our knowledge, novel type of SF remodeling. Our findings provide evidence to suggest SF sarcomeres act as strain sensors and are interconnected to support communication of mechanical information.

The actin regulator zyxin reinforces airway smooth muscle and accumulates in airways of fatal asthmatics.

Bronchospasm induced in non-asthmatic human subjects can be easily reversed by a deep inspiration (DI) whereas bronchospasm that occurs spontaneously in asthmatic subjects cannot. This physiological effect of a DI has been attributed to the manner in which a DI causes airway smooth muscle (ASM) cells to stretch, but underlying molecular mechanisms–and their failure in asthma–remain obscure. Using cells and tissues from wild type and zyxin-/- mice we report responses to a transient stretch of physiologic magnitude and duration. At the level of the cytoskeleton, zyxin facilitated repair at sites of stress fiber fragmentation. At the level of the isolated ASM cell, zyxin facilitated recovery of contractile force. Finally, at the level of the small airway embedded with a precision cut lung slice, zyxin slowed airway dilation. Thus, at each level zyxin stabilized ASM structure and contractile properties at current muscle length. Furthermore, when we examined tissue samples from humans who died as the result of an asthma attack, we found increased accumulation of zyxin compared with non-asthmatics and asthmatics who died of other causes. Together, these data suggest a biophysical role for zyxin in fatal asthma.