Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density.

Intracellular pH is a potent modulator of neuronal functions. By catalyzing (de)hydration of CO2 , intracellular carbonic anhydrase (CAi ) isoforms CA2 and CA7 contribute to neuronal pH buffering and dynamics. The presence of two highly active isoforms in neurons suggests that they may serve isozyme-specific functions unrelated to CO2 -(de)hydration. Here, we show that CA7, unlike CA2, binds to filamentous actin, and its overexpression induces formation of thick actin bundles and membrane protrusions in fibroblasts. In CA7-overexpressing neurons, CA7 is enriched in dendritic spines, which leads to aberrant spine morphology. We identified amino acids unique to CA7 that are required for direct actin interactions, promoting actin filament bundling and spine targeting. Disruption of CA7 expression in neocortical neurons leads to higher spine density due to increased proportion of small spines. Thus, our work demonstrates highly distinct subcellular expression patterns of CA7 and CA2, and a novel, structural role of CA7.

Role for formin-like 1-dependent acto-myosin assembly in lipid droplet dynamics and lipid storage.

Lipid droplets (LDs) are cellular organelles specialized in triacylglycerol (TG) storage undergoing homotypic clustering and fusion. In non-adipocytic cells with numerous LDs this is balanced by poorly understood droplet dissociation mechanisms. We identify non-muscle myosin IIa (NMIIa/MYH-9) and formin-like 1 (FMNL1) in the LD proteome. NMIIa and actin filaments concentrate around LDs, and form transient foci between dissociating LDs. NMIIa depletion results in decreased LD dissociations, enlarged LDs, decreased hydrolysis and increased storage of TGs. FMNL1 is required for actin assembly on LDs in vitro and for NMIIa recruitment to LDs in cells. We propose a novel acto-myosin structure regulating lipid storage: FMNL1-dependent assembly of myosin II-functionalized actin filaments on LDs facilitates their dissociation, thereby affecting LD surface-to-volume ratio and enzyme accessibility to TGs. In neutrophilic leucocytes from MYH9-related disease patients NMIIa inclusions are accompanied by increased lipid storage in droplets, suggesting that NMIIa dysfunction may contribute to lipid imbalance in man.

Tropomyosin Isoforms Specify Functionally Distinct Actin Filament Populations In Vitro.

Actin filaments assemble into a variety of networks to provide force for diverse cellular processes [1]. Tropomyosins are coiled-coil dimers that form head-to-tail polymers along actin filaments and regulate interactions of other proteins, including actin-depolymerizing factor (ADF)/cofilins and myosins, with actin [2-5]. In mammals, >40 tropomyosin isoforms can be generated through alternative splicing from four tropomyosin genes. Different isoforms display non-redundant functions and partially non-overlapping localization patterns, for example within the stress fiber network [6, 7]. Based on cell biological studies, it was thus proposed that tropomyosin isoforms may specify the functional properties of different actin filament populations [2]. To test this hypothesis, we analyzed the properties of actin filaments decorated by stress-fiber-associated tropomyosins (Tpm1.6, Tpm1.7, Tpm2.1, Tpm3.1, Tpm3.2, and Tpm4.2). These proteins bound F-actin with high affinity and competed with α-actinin for actin filament binding. Importantly, total internal reflection fluorescence (TIRF) microscopy of fluorescently tagged proteins revealed that most tropomyosin isoforms cannot co-polymerize with each other on actin filaments. These isoforms also bind actin with different dynamics, which correlate with their effects on actin-binding proteins. The long isoforms Tpm1.6 and Tpm1.7 displayed stable interactions with actin filaments and protected filaments from ADF/cofilin-mediated disassembly, but did not activate non-muscle myosin IIa (NMIIa). In contrast, the short isoforms Tpm3.1, Tpm3.2, and Tpm4.2 displayed rapid dynamics on actin filaments and stimulated the ATPase activity of NMIIa, but did not efficiently protect filaments from ADF/cofilin. Together, these data provide experimental evidence that tropomyosin isoforms segregate to different actin filaments and specify functional properties of distinct actin filament populations.

Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly.

Adhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells.

Cofilin-2 controls actin filament length in muscle sarcomeres.

ADF/cofilins drive cytoskeletal dynamics by promoting the disassembly of "aged" ADP-actin filaments. Mammals express several ADF/cofilin isoforms, but their specific biochemical activities and cellular functions have not been studied in detail. Here, we demonstrate that the muscle-specific isoform cofilin-2 promotes actin filament disassembly in sarcomeres to control the precise length of thin filaments in the contractile apparatus. In contrast to other isoforms, cofilin-2 efficiently binds and disassembles both ADP- and ATP/ADP-Pi-actin filaments. We mapped surface-exposed cofilin-2-specific residues required for ATP-actin binding and propose that these residues function as an "actin nucleotide-state sensor" among ADF/cofilins. The results suggest that cofilin-2 evolved specific biochemical and cellular properties that allow it to control actin dynamics in sarcomeres, where filament pointed ends may contain a mixture of ADP- and ATP/ADP-Pi-actin subunits. Our findings also offer a rationale for why cofilin-2 mutations in humans lead to myopathies.

Palladin promotes assembly of non-contractile dorsal stress fibers through VASP recruitment.

Stress fibers are major contractile actin structures in non-muscle cells where they have an important role in adhesion, morphogenesis and mechanotransduction. Palladin is a multidomain protein, which associates with stress fibers in a variety of cell types. However, the exact role of palladin in stress fiber assembly and maintenance has remained obscure, and whether it functions as an actin filament crosslinker or scaffolding protein was unknown. We demonstrate that palladin is specifically required for the assembly of non-contractile dorsal stress fibers, and is, consequently, essential for the generation of stress fiber networks and the regulation of cell morphogenesis in osteosarcoma cells migrating in a three-dimensional collagen matrix. Importantly, we reveal that palladin is necessary for the recruitment of vasodilator stimulated phosphoprotein (VASP) to dorsal stress fibers and that it promotes stress fiber assembly through VASP. Both palladin and VASP display similar rapid dynamics at dorsal stress fibers, suggesting that they associate with stress fibers as a complex. Thus, palladin functions as a dynamic scaffolding protein that promotes the assembly of dorsal stress fibers by recruiting VASP to these structures.

Evidence for a role of MRCK in mediating HeLa cell elongation induced by the C1 domain ligand HMI-1a3.

Diacylglycerol (DAG) is a central mediator of signaling pathways that regulate cell proliferation, survival and apoptosis. Therefore, C1 domain, the DAG binding site within protein kinase C (PKC) and other DAG effector proteins, is considered a potential cancer drug target. Derivatives of 5-(hydroxymethyl)isophthalic acid are a novel group of C1 domain ligands with antiproliferative and differentiation-inducing effects. Our previous work showed that these isophthalate derivatives exhibit antiproliferative and elongation-inducing effects in HeLa human cervical cancer cells. In this study we further characterized the effects of bis(3-trifluoromethylbenzyl) 5-(hydroxymethyl)isophthalate (HMI-1a3) on HeLa cell proliferation and morphology. HMI-1a3-induced cell elongation was accompanied with loss of focal adhesions and actin stress fibers, and exposure to HMI-1a3 induced a prominent relocation of cofilin-1 into the nucleus regardless of cell phenotype. The antiproliferative and morphological responses to HMI-1a3 were not modified by pharmacological inhibition or activation of PKC, or by RNAi knock-down of specific PKC isoforms, suggesting that the effects of HMI-1a3 were not mediated by PKC. Genome-wide gene expression microarray and gene set enrichment analysis suggested that, among others, HMI-1a3 induces changes in small GTPase-mediated signaling pathways. Our experiments revealed that the isophthalates bind also to the C1 domains of ß2-chimaerin, protein kinase D (PKD) and myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK), which are potential mediators of small GTPase signaling and cytoskeletal reorganization. Pharmacological inhibition of MRCK, but not that of PKD attenuated HMI-1a3-induced cell elongation, suggesting that MRCK participates in mediating the effects of HMI-1a3 on HeLa cell morphology.

Actin stress fibers--assembly, dynamics and biological roles.

Actin filaments assemble into diverse protrusive and contractile structures to provide force for a number of vital cellular processes. Stress fibers are contractile actomyosin bundles found in many cultured non-muscle cells, where they have a central role in cell adhesion and morphogenesis. Focal-adhesion-anchored stress fibers also have an important role in mechanotransduction. In animal tissues, stress fibers are especially abundant in endothelial cells, myofibroblasts and epithelial cells. Importantly, recent live-cell imaging studies have provided new information regarding the mechanisms of stress fiber assembly and how their contractility is regulated in cells. In addition, these studies might elucidate the general mechanisms by which contractile actomyosin arrays, including muscle cell myofibrils and cytokinetic contractile ring, can be generated in cells. In this Commentary, we discuss recent findings concerning the physiological roles of stress fibers and the mechanism by which these structures are generated in cells.

A molecular pathway for myosin II recruitment to stress fibers.

BACKGROUND: Cell migration and morphogenesis are driven by both protrusive and contractile actin filament structures. The assembly mechanisms of lamellipodial and filopodial actin filament arrays, which provide the force for plasma membrane protrusions through actin filament treadmilling, are relatively well understood. In contrast, the mechanisms by which contractile actomyosin arrays such as stress fibers are generated in cells, and how myosin II is specifically recruited to these structures, are not known. RESULTS: We demonstrate that four functionally distinct tropomyosins are required for assembly of stress fibers in cultured osteosarcoma cells. Tm1, Tm2/3, and Tm5NM1/2 stabilize actin filaments at distinct stress fiber regions. In contrast, Tm4 promotes stress fiber assembly by recruiting myosin II to stress fiber precursors. Elimination of any one of the tropomyosins fatally compromises stress fiber formation. Importantly, Dia2 formin is critical to stress fiber assembly by nucleating Tm4-decorated actin filaments at the cell cortex. Myosin II is specifically recruited through a Tm4-dependent mechanism to the Dia2-nucleated filaments, which subsequently assemble endwise with Arp2/3-nucleated actin filament structures to yield contractile stress fibers. CONCLUSIONS: These experiments identified a pathway, involving Dia2- and Arp2/3-promoted actin filament nucleation and several functionally distinct tropomyosins, that is required for generation of contractile actomyosin arrays in cells.