Requirement of Drosophila I(2)gl function for survival of the germline cells and organization of the follicle cells in a columnar epithelium during oogenesis.

The lethal(2)giant larvae gene, or 1(2)gl, encodes a widely expressed cytoskeletal protein which acts in numerous biological processes during embryogenesis and oogenesis, including cell proliferation, and morphogenetic movements. Having identified the nucleotide change occurring in the l(2)gl(ts3) sequence, we produced by site-directed mutagenesis the identical change leading to the substitution of a serine by a phenylalanine at position 311 of p127l(2)gl and introduced the modified l(2)glF311 gene into l(2)gl flies. The transgene can fully rescue the development of l(2)gl flies raised at 22 degrees C but causes drastic effects on their development at 29 degrees C confirming the temperature sensitivity of the phenylalanine substitution at position 311. Fertility of females, albeit not of males, was strongly affected. Temperature-shift experiments and microscopic examination of ovaries showed that the mutation blocked egg chamber development at the onset of vitellogenesis (stages 8-9) with growth arrest of the oocyte, incomplete follicle cell migration over the oocyte associated with abnormal organization of the follicular epithelium, and apoptosis of the germline cells, as measured by TUNEL assays. By comparison to wildtype, we found that p127F311 is already reduced in amount at 22 degrees C and delocalized from the cytoskeletal matrix, albeit without affecting the apical localization of myosin II, a major partner of p127. At 29 degrees C, the level of p127F311 is even more reduced and the distribution of myosin-II becomes markedly altered at the apices of the follicle cells. These data indicate that during oogenesis p127 plays a critical function at the onset of vitellogenesis and regulates growth of the oocyte, follicle cell migration over the oocyte and their organization in a palisadic epithelium, as well as viability of the germline cells.     

The Drosophila lethal(2)giant larvae tumor suppressor protein is a component of the cytoskeleton

Tumor suppressor genes act as recessive determinants of cancer. In Drosophila these genes play a role in normal development and are essential for regulating cell growth and differentiation. Mutations in the gene, lethal(2)giant larvae, l(2)gl, besides causing malignant tumors in the brain and imaginal discs, generate developmental defects in a number of other tissues. Much of the uncertainty regarding the function of the l(2)gl gene product, p127, results from a lack of knowledge as to the precise location of this protein in the cell. We have investigated the cellular and subcellular localization of p127, using confocal and electron microscopy as well as biochemical and cell fractionation procedures. Our analyses indicate that p127 is located entirely within the cell in both the cytoplasm and bound to the inner face of lateral cell membranes in regions of cell junctions. On the membrane, p127 can form large aggregates which are resistant to solubilization by nonionic detergents, indicating that p127 is participating in a cytoskeletal matrix. These findings suggest that the changes in cell shape and the loss of apical-basal polarity observed in tumorous tissues are a direct result of alterations in the cytoskeleton organization caused by l(2)gl inactivation and also suggest that p127 is involved in a cytoskeletal-based intercellular communication system directing cell differentiation.     

Induced paternal effects mimic cytoplasmic incompatibility in Drosophila

Wolbachia is an intracellular microbe found in a wide diversity of arthropod and filarial nematode hosts. In arthropods these common bacteria are reproductive parasites that manipulate central elements of their host's reproduction to increase their own maternal transmission in one of several ways. Cytoplasmic incompatibility (CI) is one such manipulation where sperm are somehow modified in infected males and this modification must be rescued by the presence of the same bacterial strain in the egg for normal development to proceed. The molecular mechanisms involved in the expression of CI are unknown. Here we show that Wolbachia infection results in increased mRNA and protein expression of the Drosophila simulans nonmuscle myosin II gene zipper. Induced overexpression of zipper in Wolbachia-free transgenic D. melanogaster males results in paternal-effect lethality that mimics the fertilization defects associated with CI. Likewise, overexpression of the tumor suppressor gene, lethal giant larvae [l(2)gl], results in egg lethality and a CI phenotype. Stoichiometric levels of zipper and l(2)gl are required for proper segregation of cellular determinants during neuroblast stem cell division. Taken together these results form the basis of a working hypothesis whereby Wolbachia induces paternal effects in sperm by manipulating the expression of key regulators of cytoskeletal activity during spermatogenesis.     

The timing of drosophila salivary gland apoptosis displays an l(2)gl-dose response

During Drosophila metamorphosis, larval tissues, such as the salivary glands, are histolysed whereas imaginal tissues differentiate into adult structures forming at eclosion a fly-shaped adult. Inactivation of the lethal(2)giant larvae (l(2)gl) gene encoding the cytoskeletal associated p127 protein, causes malignant transformation of brain neuroblasts and imaginal disc cells with developmental arrest at the larval-pupal transition phase. At this stage, p127 is expressed in wild-type salivary glands which become fully histolysed 12 - 13 h after pupariation. By contrast to wild-type, administration of 20-hydroxyecdsone to l(2)gl-deficient salivary glands is unable to induce histolysis, although it releases stored glue granules and gives rise to a nearly normal pupariation chromosome puffing, indicating that p127 is required for salivary gland apoptosis. To unravel the l(2)gl function in this tissue we used transgenic lines expressing reduced ( approximately 0.1) or increased levels of p127 (3.0). Here we show that the timing of salivary gland histolysis displays an l(2)gl-dose response. Reduced p127 expression delays histolysis whereas overexpression accelerates this process without affecting the duration of third larval instar, prepupal and pupal development. Similar l(2)gl-dependence is noticed in the timing of expression of the cell death genes reaper, head involution defective and grim, supporting the idea that p127 plays a critical role in the implementation of ecdysone-triggered apoptosis. These experiments show also that the timing of salivary gland apoptosis can be manipulated without affecting normal development and provide ways to investigate the nature of the components specifically involved in the apoptotic pathway of the salivary glands.     

Structure of the l(2)gl gene of Drosophila and delimitation of its tumor suppressor domain

We have previously cloned lethal(2)giant larvae, a tumor-suppressor gene of Drosophila that normally controls cell proliferation and/or differentiation in the optic centers of the brain and the imaginal discs. Here we describe the structure of the l(2)gl genes as determined by sequencing genomic and cDNA clones. The structure of the cDNAs indicates the use of alternative splicing, either in the 5' untranslated exons or in the 3' coding exons. Thus the gene encodes two putative proteins of 1161 and 708 amino acids, p127 and p78, respectively, differing at their C termini. A 3'-truncated l(2)gl transposon that leaves the coding sequence of p78 intact but deletes 141 residues of p127 was capable of suppressing tumor formation in l(2)gl-deficient animals. These results suggest that the putative p78 protein is effective in controlling cell proliferation and/or differentiation.     

Detection of a specific isoform of alpha-actinin with antisera directed against dystrophin

We have characterized a protein immunologically related to dystrophin, the protein product of the Duchenne muscular dystrophy gene. We identify this related protein as a fast-twitch glycolytic isoform (mouse extensor digitorum longus-specific) of myofibrillar alpha-actinin. This specific isoform of alpha-actinin exhibits a more restricted pattern of expression in skeletal muscle than fast-twitch-specific isoforms of both myosin and Ca2+-ATPase. Our results provide evidence that dystrophin and myofibrillar alpha-actinin are related proteins, reinforcing the previous data concerning the sequence homologies noted between nonmuscle cytoskeletal alpha-actinin and dystrophin. In addition, we describe the first antisera directed against a specific myofibrillar skeletal muscle isoform of alpha-actinin.     

Gene duplication and conversion events shaped three homologous, differentially expressed myosin regulatory light chain (MLC2) genes

Myosin II is a hexameric protein complex consisting of two myosin heavy chains, two myosin essential light chains and two myosin regulatory light chains. Multiple subunit isoforms exist, allowing great diversity in myosin II composition which likely impacts on its contractile properties. Little is known about the evolutionary origin, expression pattern and function of myosin regulatory light chain (MLC2) isoforms. We analysed the evolutionary relationship between smooth muscle (sm), nonmuscle (nm) and nonmuscle-like (nml) MLC2 genes, which encode three homologous proteins expressed in nonmuscle cells. The three genes arose by successive gene duplication events. The high sequence similarity between the tandemly arranged nm- and nml-MLC2 genes is best explained by gene conversion. Urea/glycerol-polyacrylamide gel electrophoresis and RNA analysis were employed to monitor expression of sm-, nm- and nml-MLC2 in human and mouse cell lines. Conspicuous differences between transformed and non-transformed cells were observed, with sm-MLC2 being suppressed in Ras-transformed cells. Our findings shed light on the evolutionary history of three homologous MLC2 proteins and point to isoform-specific cell growth-related roles in nonmuscle cell myosin II contractility.     

F-actin and myosin II binding domains in supervillin

Detergent-resistant membranes contain signaling and integral membrane proteins that organize cholesterol-rich domains called lipid rafts. A subset of these detergent-resistant membranes (DRM-H) exhibits a higher buoyant density ( approximately 1.16 g/ml) because of association with membrane skeleton proteins, including actin, myosin II, myosin 1G, fodrin, and an actin- and membrane-binding protein called supervillin (Nebl, T., Pestonjamasp, K. N., Leszyk, J. D., Crowley, J. L., Oh, S. W., and Luna, E. J. (2002) J. Biol. Chem. 277, 43399-43409). To characterize interactions among DRM-H cytoskeletal proteins, we investigated the binding partners of the novel supervillin N terminus, specifically amino acids 1-830. We find that the supervillin N terminus binds directly to myosin II, as well as to F-actin. Three F-actin-binding sites were mapped to sequences within amino acids approximately 280-342, approximately 344-422, and approximately 700-830. Sequences with combinations of these sites promote F-actin cross-linking and/or bundling. Supervillin amino acids 1-174 specifically interact with the S2 domain in chicken gizzard myosin and nonmuscle myosin IIA (MYH-9) but exhibit little binding to skeletal muscle myosin II. Direct or indirect binding to filamin also was observed. Overexpression of supervillin amino acids 1-174 in COS7 cells disrupted the localization of myosin IIB without obviously affecting actin filaments. Taken together, these results suggest that supervillin may mediate actin and myosin II filament organization at cholesterol-rich membrane domains.     

A Novel Myosin-like Protein (Myocilin) Expressed in the Connecting Cilium of the Photoreceptor: Molecular Cloning, Tissue Expression, and Chromosomal Mapping

We have isolated a human cDNA clone encoding a novel acidic protein of MW 55,000 that we designated “myocilin” since it has homology to myosin and is localized preferentially in the ciliary rootlet and basal body of the connecting cilium of photoreceptor cells. The deduced amino acid sequence of human myocilin showed significant homologies with nonmuscle myosin ofDictyostelium discoideumin the N-terminal region and also with olfactomedin of bullfrog in the C-terminal region. Myocilin contained a leucine zipper-like motif similar to that seen in kinectin and other cytoskeletal proteins. These findings suggest that myocilin is a novel cytoskeletal protein involved in the morphogenesis of ciliated neuroepithelium such as photoreceptor cells. The myocilin gene (MYOC) was mapped to human chromosome 1q23–q24 by fluorescencein situhybridization.     

Myosin light chain kinase colocalizes with nonmuscle myosin IIB in myofibril precursors and sarcomeric Z-lines of cardiomyocytes

Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility involving actin and myosin II. MLCK is widely present in vertebrate tissues including the myocardium. However, the role of MLCK in cardiomyocyte function is not known. Previous attempts to gain insight into possible roles and identify potential molecular partners were disappointing and equivocal due to cross reactivity of early antibodies with striated muscle MLCK, which has a different genetic locus and a divergent amino acid sequence from the above mentioned enzyme. Using an immunofluorescence approach and a panel of antibodies directed against MLCK, cytoskeletal, and sarcomeric proteins, we localized MLCK to myofibril precursors and Z-lines of sarcomeres in embryonic and adult cardiomyocytes. The same structures contained nonmuscle myosin IIB implicating this protein as a possible target of MLCK. Our results suggest a role for MLCK in cardiomyocyte differentiation and contraction through regulation of nonmuscle myosin IIB.     

Metastasis-associated protein Mts1 (S100A4) inhibits CK2-mediated phosphorylation and self-assembly of the heavy chain of nonmuscle myosin

A role for EF-hand calcium-binding protein Mts1 (S100A4) in the phosphorylation and the assembly of myosin filaments was studied. The nonmuscle myosin molecules form bipolar filaments, which interact with actin filaments to produce a contractile force. Phosphorylation of the myosin plays a regulatory role in the myosin assembly. In the presence of calcium, Mts1 binds at the C-terminal end of the myosin heavy chain close to the site of phosphorylation by protein kinase CK2 (Ser1944). In the present study, we have shown that interaction of Mts1 with the human platelet myosin or C-terminal fragment of the myosin heavy chain inhibits phosphorylation of the myosin heavy chain by protein kinase CK2 in vitro. Mts1 might also bind directly the beta subunit of protein kinase CK2, thereby modifying the enzyme activity. Our results indicate that myosin oligomers were disassembled in the presence of Mts1. The short C-terminal fragment of the myosin heavy chain was totally soluble in the presence of an equimolar amount of Mts1 at low ionic conditions (50 mM NaCl). Depolymerization was found to be calcium-dependent and could be blocked by EGTA. Our data suggest that Mts1 can increase myosin solubility and therefore suppress its assembly.     

Expression of green or red fluorescent protein (GFP or DsRed) linked proteins in nonmuscle and muscle cells

The introduction of the green fluorescent protein (GFP) plasmids that allow proteins and peptides to be expressed with a fluorescent tag has had a major impact on the field of cell biology. It has enabled the dynamics of a wide variety of proteins to be analyzed that could not otherwise be detected in live cells. Transient transfections of muscle and nonmuscle cells with plasmids encoding various cytoskeletal proteins ligated to green fluorescent protein or Ds red protein allow changes in the cytoskeletal network to be studied in the same cell for time periods up to several days. With this approach, proteins that could not be purified and directly labeled with fluorescent dyes and microinjected into cells can now be expressed and visualized in a wide variety of cells. Procedures are presented for transfection of the nonmuscle cell, PtK2, and primary cultures of embryonic chick myocytes, and for studying the live transfected cells.     

Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.     

Reorganization of structural proteins in vascular smooth muscle cells grown in collagen gel and basement membrane matrices (Matrigel): a comparison with their in situ counterparts

When smooth muscle cells are enzyme-dispersed from tissues they lose their original filament architecture and extracellular matrix surrounds. They then reorganize their structural proteins to accommodate a 2-D growth environment when seeded onto culture dishes. The aim of the present study was to determine the expression and reorganization of the structural proteins in rabbit aortic smooth muscle cells seeded into 3-D collagen gel and Matrigel (a basement membrane matrix). It was shown that smooth muscle cells seeded in both gels gradually reorganize their structural proteins into an architecture similar to that of their in vivo counterparts. At the same time, a gradual decrease in levels of smooth muscle-specific contractile proteins (mainly smooth muscle myosin heavy chain-2) and an increase in beta-nonmuscle actin occur, independent of both cell growth and extracellular matrix components. Thus, smooth muscle cells in 3-D extracellular matrix culture and in vivo have a similar filament architecture in which the contractile proteins such as actin, myosin, and alpha-actinin are organized into longitudinally arranged "myofibrils" and the vimentin-containing intermediate filaments form a meshed cytoskeletal network. However, the myofibrils reorganized in vitro contain less smooth muscle-specific and more nonmuscle contractile proteins.     

Genetic analysis demonstrates a direct link between rho signaling and nonmuscle myosin function during Drosophila morphogenesis

A dynamic actomyosin cytoskeleton drives many morphogenetic events. Conventional nonmuscle myosin-II (myosin) is a key chemomechanical motor that drives contraction of the actin cytoskeleton. We have explored the regulation of myosin activity by performing genetic screens to identify gene products that collaborate with myosin during Drosophila morphogenesis. Specifically, we screened for second-site noncomplementors of a mutation in the zipper gene that encodes the nonmuscle myosin-II heavy chain. We determined that a single missense mutation in the zipper(Ebr) allele gives rise to its sensitivity to second-site noncomplementation. We then identify the Rho signal transduction pathway as necessary for proper myosin function. First we show that a lethal P-element insertion interacts genetically with zipper. Subsequently we show that this second-site noncomplementing mutation disrupts the RhoGEF2 locus. Next, we show that two EMS-induced mutations, previously shown to interact genetically with zipper(Ebr), disrupt the RhoA locus. Further, we have identified their molecular lesions and determined that disruption of the carboxyl-terminal CaaX box gives rise to their mutant phenotype. Finally, we show that RhoA mutations themselves can be utilized in genetic screens. Biochemical and cell culture analyses suggest that Rho signal transduction regulates the activity of myosin. Our studies provide direct genetic proof of the biological relevance of regulation of myosin by Rho signal transduction in an intact metazoan.     

The essential role of PP1beta in Drosophila is to regulate nonmuscle myosin

Reversible phosphorylation of myosin regulatory light chain (MRLC) is a key regulatory mechanism controlling myosin activity and thus regulating the actin/myosin cytoskeleton. We show that Drosophila PP1beta, a specific isoform of serine/threonine protein phosphatase 1 (PP1), regulates nonmuscle myosin and that this is the essential role of PP1beta. Loss of PP1beta leads to increased levels of phosphorylated nonmuscle MRLC (Sqh) and actin disorganisation; these phenotypes can be suppressed by reducing the amount of active myosin. Drosophila has two nonmuscle myosin targeting subunits, one of which (MYPT-75D) resembles MYPT3, binds specifically to PP1beta, and activates PP1beta's Sqh phosphatase activity. Expression of a mutant form of MYPT-75D that is unable to bind PP1 results in elevation of Sqh phosphorylation in vivo and leads to phenotypes that can also be suppressed by reducing the amount of active myosin. The similarity between fly and human PP1beta and MYPT genes suggests this role may be conserved.