Zasp is required for the assembly of functional integrin adhesion sites

The integrin family of heterodimeric transmembrane receptors mediates cell–matrix adhesion. Integrins often localize in highly organized structures, such as focal adhesions in tissue culture and myotendinous junctions in muscles. Our RNA interference screen for genes that prevent integrin-dependent cell spreading identifies Z band alternatively spliced PDZ-motif protein (zasp), encoding the only known Drosophila melanogaster Alp/Enigma PDZ-LIM domain protein. Zasp localizes to integrin adhesion sites and its depletion disrupts integrin adhesion sites. In tissues, Zasp colocalizes with βPS integrin in myotendinous junctions and with α-actinin in muscle Z lines. Zasp also physically interacts with α-actinin. Fly larvae lacking Zasp do not form Z lines and fail to recruit α-actinin to the Z line. At the myotendinous junction, muscles detach in zasp mutants with the onset of contractility. Finally, Zasp interacts genetically with integrins, showing that it regulates integrin function. Our observations point to an important function for Zasp in the assembly of integrin adhesion sites both in cell culture and in tissues

Comparative RNAi screening identifies a conserved core metazoan actinome by phenotype

RNAi Screens in Drosophila and human cells for novel actin regulators revealed conserved roles for proteins involved in nuclear actin export, RNA splicing, and ubiquitination

Retraction of the Drosophila germ band requires cell–matrix interaction

Integrins and laminins are important mediators of cell–matrix interactions in both vertebrates and invertebrates. Here, we show that germ-band retraction in the Drosophila embryo, during which the tail end of the embryo retracts to its final posterior position, allows the investigation of cell spreading and lamellipodia formation in real time in vivo. We demonstrate that α1, 2 laminin and αPS3βPS integrin are required for the spreading of a small group of cells of the amnioserosa epithelium over the tail end of the germ band. We further implicate a role for this spreading in the process of germ-band retraction

Filamin actin-binding and titin-binding fulfill distinct functions in Z-disc cohesion

Many proteins contribute to the contractile properties of muscles, most notably myosin thickfilaments, which are anchored at the M-line, and actin thin filaments, which are anchored atthe Z-discs that border each sarcomere. In humans, mutations in the actin-binding proteinFilamin-C result in myopathies, but the underlying molecular function is not well understood.Here we show using Drosophila indirect flight muscle that the filamin ortholog Cheerio inconjunction with the giant elastic protein titin plays a crucial role in keeping thin filaments stablyanchored at the Z-disc. We identify the filamin domains required for interaction with thetitin ortholog Sallimus, and we demonstrate a genetic interaction of filamin with titin andactin. Filamin mutants disrupting the actin- or the titin-binding domain display distinct phenotypes,with Z-discs breaking up in parallel or perpendicularly to the myofibril, respectively.Thus, Z-discs require filamin to withstand the strong contractile forces acting on them

Characterizing the actin-binding ability of Zasp52 and its contribution to myofibril assembly.

In sarcomeres, α-actinin crosslinks thin filaments and anchors them at the Z-disc. Drosophila melanogaster Zasp52 also localizes at Z-discs and interacts with α-actinin via its extended PDZ domain, thereby contributing to myofibril assembly and maintenance, yet the detailed mechanism of Zasp52 function is unknown. Here we show a strong genetic interaction between actin and Zasp52 during indirect flight muscle assembly, indicating that this interaction plays a critical role during myofibril assembly. Our results suggest that Zasp52 contains an actin-binding site, which includes the extended PDZ domain and the ZM region. Zasp52 binds with micromolar affinity to monomeric actin. A co-sedimentation assay indicates that Zasp52 can also bind to F-actin. Finally, we use in vivo rescue assays of myofibril assembly to show that the α-actinin-binding domain of Zasp52 is not sufficient for a full rescue of Zasp52 mutants suggesting additional contributions of Zasp52 actin-binding to myofibril assembly

Alp/Enigma family proteins cooperate in Z-disc formation and myofibril assembly.

The Drosophila Alp/Enigma family protein Zasp52 localizes to myotendinous junctions and Z-discs. It is required for terminal muscle differentiation and muscle attachment. Its vertebrate ortholog ZASP/Cypher also localizes to Z-discs, interacts with α-actinin through its PDZ domain, and is involved in Z-disc maintenance. Human mutations in ZASP cause myopathies and cardiomyopathies. Here we show that Drosophila Zasp52 is one of the earliest markers of Z-disc assembly, and we use a Zasp52-GFP fusion to document myofibril assembly by live imaging. We demonstrate that Zasp52 is required for adult Z-disc stability and pupal myofibril assembly. In addition, we show that two closely related proteins, Zasp66 and the newly identified Zasp67, are also required for adult Z-disc stability and are participating with Zasp52 in Z-disc assembly resulting in more severe, synergistic myofibril defects in double mutants. Zasp52 and Zasp66 directly bind to α-actinin, and they can also form a ternary complex. Our results indicate that Alp/Enigma family members cooperate in Z-disc assembly and myofibril formation; and we propose, based on sequence analysis, a novel class of PDZ domain likely involved in α-actinin binding

mBtd is required to maintain signaling during murine limb development

buttonhead (btd) encodes an SP1-like transcription factor required for the generation and specification of Drosophila head segments. We identified a murine btd homolog, termed mouse Btd (mBtd), which can support btd-dependent head development in transgenic fly embryos. Functional studies show that mBtd-deficient mice develop to term and die at birth. They exhibit brain malformations, posterior axial skeleton truncations, and shortened limbs. We present evidence that mBtd is required during early limb development to maintain, but not to initiate Wnt/β-catenin-dependent FGF, Shh, and BMP-mediated signaling. The data indicate that mBtd represents a novel key player mediating proximodistal outgrowth of the limb

Cher localizes to the Z-disc in the IFM and is necessary for sarcomere structural stability.

<p><b>(A</b>) <i>cher</i> genomic region showing several transcripts and several protein isoforms that can be grouped based on their molecular weight into 4 groups (CherA-D). Green triangles represent transgenic insertion sites of protein-trap lines, each bearing a splice acceptor site followed by a Venus and a Flag tag. The protein domains encoded by different exons are indicated at the bottom: CH (ABD), calponin homology domain 1 and 2 comprises the actin-binding domain; Ig, immunoglobulin-like domains; DD, dimerization domain. Asterisks denote location of the premature stop point mutants in <i>cher</i>^{<i>Q1042x</i>} and <i>cher</i>^{<i>Q1415sd</i>}. The <i>cher</i>^<i>Δ5</i> deleted region is denoted by a black line (<b>B</b>) Immunoblot from <i>cher</i>^{<i>CPTI1399</i>} thoraces incubated with anti-Flag antibody reveals 4 Cher isoforms, specifically depleted in <i>Mef2-Gal4</i>>UAS-<i>cher-JF</i> RNAi. (<b>C, D</b>) IFM confocal images from heterozygous Flag-tagged Cher traps stained with anti-Flag antibody to visualize the tagged isoforms. In all images, asterisks mark Z-discs of a selected myofibril immediately above the asterisks. (<b>C</b>) All Cher isoforms, revealed by the <i>cher</i>^{<i>CPTI1403</i>} protein trap, localize at the Z-disc. (<b>D</b>) Long Cher isoforms tagged by <i>cher</i>^{<i>CPTI1399</i>} localize to the Z-disc. (<b>E</b>) Quantification of recognizable sarcomeres in control, Cher-depleted, and <i>cher</i> mutant IFM. Statistical significance assessed by one-way ANOVA with post hoc Tukey: n.s. = not significant, ** = P ≤ 0.01, **** = P ≤ 0.0001. (<b>F</b>, <b>G</b>) Confocal images from control and Cher-depleted IFM, stained with anti-Kettin antibody to visualize Z-discs in red and phalloidin to visualize actin thin filaments in green. (<b>F</b>) Regular sarcomeric structure in <i>Mef2-Gal4</i> control flies, (<b>G</b>) Depletion of Cher in <i>Mef2-Gal4</i>, <i>UAS-cher-JF</i> flies results in severe sarcomeric disorganization. Arrowheads indicate reduced actin staining at the Z-disc. Scale bars: 5 μm.</p

Overexpression defects of Zasp52 transgenes are dosage-dependent.

<p>(<b>A</b>) Western Blot visualizing UAS-Zasp52-PRΔPDZ driven by Mef2-Gal4 or UH3-Gal4. α-tubulin serves as loading control. (<b>B</b>) Confocal microscopy of IFM stained with anti-Flag antibody visualizing the transgene in green, anti-α-actinin antibody to label Z-discs in purple, as well as phalloidin to visualize actin thin filaments in red. Weak UH3-Gal4-mediated expression of UAS-Zasp52-PRΔPDZ reduces overexpression defects. Scale bar, 10 μm.</p