Soluble membrane-type 3 matrix metalloprioteinase causes changes in gene expression and increased gelatinase activity during Xenopus laevis development

Matrix metalloproteinases (MMPs) are a family of endopeptidases that cleave and remodel the extracellular matrix (ECM). Membrane-type 3 MMP (MT3-MMP) is a membrane-anchored MMP, which has recently been shown to 'shed' from the cell surface in a soluble form upon proteolytic cleavage. Shed MT-MMPs can activate gelatinase-A in vitro and have been directly linked to the metastatic potential of many cancers. Here we examined the effect of ectopic expression of full-length tethered and shed (soluble) forms of MT3-MMP during Xenopus laevis development. Injection of mRNA coding for full-length tethered MT3-MMP resulted in the delayed onset of gastrulation and subsequent defects. Phenotype severity and the frequency of embryo death were dose-dependent. Dose-dependent defects were also observed with the injection of mRNA of the soluble form, but the phenotypes and frequencies of death were greater. Histological analysis of injected embryos demonstrated defects in the organization of axial structures, such as the neural tube and somites. Embryos injected with full-length MT3-MMP mRNA showed no significant changes in expression levels of the tissue specific genes endodermin, chordin and muscle actin when examined by semi-quantitative RT-PCR. In contrast, embryos injected with the soluble form of MT3-MMP exhibited decreased expression of these same marker genes. In addition, while full-length tethered MT3-MMP failed to alter gelatinase activity, a 50% increase was measured in response to injection of the soluble form, suggesting that the two forms of this protein could play distinct roles during embryogenesis.     

A non-canonical mode of microtubule organization operates throughout pre-implantation development in mouse

In dividing animal cells, the centrosome, comprising centrioles and surrounding pericentriolar-material (PCM), is the major interphase microtubule-organizing center (MTOC), arranging a polarized array of microtubules (MTs) that controls cellular architecture. The mouse embryo is a unique setting for investigating the role of centrosomes in MT organization, since the early embryo is acentrosomal, and centrosomes emerge de novo during early cleavages. Here we use embryos from a GFP::CETN2 transgenic mouse to observe the emergence of centrosomes and centrioles in embryos, and show that unfocused acentriolar centrosomes first form in morulae (~16–32-cell stage) and become focused at the blastocyst stage (~64–128 cells) concomitant with the emergence of centrioles. We then used high-resolution microscopy and dynamic tracking of MT growth events in live embryos to examine the impact of centrosome emergence upon interphase MT dynamics. We report that pre-implantation mouse embryos of all stages employ a non-canonical mode of MT organization that generates a complex array of randomly oriented MTs that are preferentially nucleated adjacent to nuclear and plasmalemmal membranes and cell-cell interfaces. Surprisingly, however, cells of the early embryo continue to employ this mode of interphase MT organization even after the emergence of centrosomes. Centrosomes are found at MT-sparse sites and have no detectable impact upon interphase MT dynamics. To our knowledge, the early embryo is unique among proliferating cells in adopting an acentrosomal mode of MT organization despite the presence of centrosomes, revealing that the transition to a canonical mode of interphase MT organization remains incomplete prior to implantation.     

The kinetically dominant assembly pathway for centrosomal asters in Caenorhabditis elegans is gamma-tubulin dependent

gamma-Tubulin-containing complexes are thought to nucleate and anchor centrosomal microtubules (MTs). Surprisingly, a recent study (Strome, S., J. Powers, M. Dunn, K. Reese, C.J. Malone, J. White, G. Seydoux, and W. Saxton. Mol. Biol. Cell. 12:1751-1764) showed that centrosomal asters form in Caenorhabditis elegans embryos depleted of gamma-tubulin by RNA-mediated interference (RNAi). Here, we investigate the nucleation and organization of centrosomal MT asters in C. elegans embryos severely compromised for gamma-tubulin function. We characterize embryos depleted of approximately 98% centrosomal gamma-tubulin by RNAi, embryos expressing a mutant form of gamma-tubulin, and embryos depleted of a gamma-tubulin-associated protein, CeGrip-1. In all cases, centrosomal asters fail to form during interphase but assemble as embryos enter mitosis. The formation of these mitotic asters does not require ZYG-9, a centrosomal MT-associated protein, or cytoplasmic dynein, a minus end-directed motor that contributes to self-organization of mitotic asters in other organisms. By kinetically monitoring MT regrowth from cold-treated mitotic centrosomes in vivo, we show that centrosomal nucleating activity is severely compromised by gamma-tubulin depletion. Thus, although unknown mechanisms can support partial assembly of mitotic centrosomal asters, gamma-tubulin is the kinetically dominant centrosomal MT nucleator.     

Comparison of the incidence of 5-azacytidine-induced exencephaly between MT/HokIdr and Slc:ICR mice.

The incidence of 5-azacytidine-induced exencephaly was compared between MT/HokIdr strain (MT) and Slc:ICR strain (ICR) mice. MT mice have a genetic predisposition for exencephaly, but ICR mice do not. Pregnant mice were given 5-azacytidine (1 mg/kg to 100 micrograms/kg) injected intraperitoneally on Day 7.5 of gestation (vaginal plug day = Day 0.5), and fetuses were observed for external malformations on Day 18.5 of gestation. One hundred micrograms/kg 5-azacytidine induced exencephaly in MT mice but not in ICR mice, and 1 mg/kg 5-azacytidine resulted in resorptions in MT mice but caused exencephaly in ICR mice. These results indicated that MT mice had 10-fold more sensitivity to 5-azacytidine than ICR mice. It seems likely that less than effective doses of teratogens for animals without genetic predispositions are still effective in inducing malformations in animals with a genetic predisposition for malformations. When 4-somite-stage embryos of both MT and ICR mice were cultured in rat serum supplemented with 5-azacytidine, 0.02 micrograms/ml 5-azacytidine induced the failure of closure of cephalic neural tube in MT embryos but not in ICR embryos, and 0.2 micrograms/ml 5-azacytidine induced severe growth retardation in MT embryos but in ICR embryos it only induced embryos with smaller heads and fewer somites than in control. These results indicated that MT mouse embryos in culture also had a 10-fold-increased sensitivity to 5-azacytidine compared with ICR mouse embryos, suggesting maternal effects play no significant role in their increased sensitivity to 5-azacytidine.     

Anatomical studies on seeds and seedlings of some Utricularia (Lentibulariaceae)

The undifferentiated mature embryos of someUtricularia studied consist of large polygonal cells which contain many starch grains. Later, they consist mostly of large polygonal cells with some small cells in one or more regions adjacent to the seed coat, which lack starch grains but have rather easily visible nuclei. After these small cells divide and produce more cells of the same type toward the midcenter of the embryo, they form one or more primordia which grow into the primary structures of the seedling. The primordia of the embryo seem to have no pattern as to which primary vegetative structure they will develop into. However, the terrestrial species ofUtricularia studied showed some non-cotyledonous, primary differences in germination patterns; some form primary foliar units, some form stolons, and some form bladders. The first and second primary foliar units of aquaticUtricularia radiata andV. gibba subsp.gibba do not follow in the same manner as cotyledons. No cotyledon could be distinguished in any of theUtricularia seedlings studied. In the seedlings ofU. gibba subsp.gibba andU. radiata, appearance and differentiation of the vascular elements occur simultaneously in more than one direction, toward the meristematic tissues of primary vegetative structures and toward the midcenter of the embryos. Lloyd’s proposed term: “cotyledonoids” (1942) and that of Kumazawa: “cotyledons” (1967) should not be used for the first and second primary foliar units in either aquatic or terrestrial to terrestrial-epiphyticUtricularia, unless further supportive evidence is accumulated.     

Changes in zinc,copper and metallothionein contents during oocyte growth and early development of the teleost Danio rerio (zebrafish)

In the present report, we investigated zinc, copper and metallothionein (MT) contents in zebrafish oocytes and embryos. Our results demonstrate that the metal content increases during oocytes maturation. Zinc increases from 30 ng/oocyte (stage-1 oocytes) to 100 ng/oocyte (stage-3 oocytes); copper varied from 1 ng/oocyte (stage-1 oocytes) to 3.5 ng/oocyte (stage-3 oocytes). During embryogenesis, zinc and copper contents dramatically increase after fertilisation around the 512-cells stage, then slowly decrease until the mid-gastrula stage. During oocyte growth, the changes in the MT level are proportional to metal content, whereas during embryogenesis the pattern of MT accumulation does not parallel that of the two metals. Indeed, the maternal pool of MT decreases steadily during the early stages of the development until the gastrula stage. We have examined the effect of cadmium on the expression of MT during zebrafish development. After cadmium exposure, MT content increases in embryos at the blastula stage, whereas no induction occurs in embryos at the gastrula stage. However, pre-treatment of embryos at the gastrula stage with 5-aza-2'-deoxycytidine induces MT synthesis following exposure to cadmium. These observations show that changes in metal levels are not correlated to MT content in the embryo, whereas DNA methylation is one of the factors regulating MT expression.     

Three-dimensional solution structure of mouse [Cd7]-metallothionein-1 by homonuclear and heteronuclear NMR spectroscopy

Sequential 1H-NMR assignments of mouse [Cd7]-metallothionein-1 (MT1) have been carried out by standard homonuclear NMR methods and the use of an accordion-heteronuclear multiple quantum correlation (HMQC) experiment for establishing the metal, 113Cd2+, to cysteine connectivities. The three-dimensional structure was then calculated using the distance constraints from two-dimensional nuclear Overhauser effect (NOE) spectroscopy spectra and the Cys-Cd connectivities as input for a distance geometry-dynamical simulated annealing protocol in X-PLOR 3.851. Similar to the mammalian MT2 isoforms, the homologous primary structure of MT1 suggested two separate domains, each containing one metal cluster. Because there were no interdomain constraints, the structure calculation for the N-terminal beta- and the C-terminal alpha-domain were carried out separately. The structures are based on 409 NMR constraints, consisting of 381 NOEs and 28 cysteine-metal connectivities. The only elements of regular secondary structure found were two short stretches of 3(10) helices along with some half-turns in the alpha-domain. Structural comparison with rat liver MT2 showed high similarity, with the beta-domain structure in mouse MT1 showing evidence of increased flexibility compared to the same domain in MT2. The latter was reflected by the presence of fewer interresidue NOEs, no slowly exchanging backbone amide protons, and enhanced cadmium-cadmium exchange rates found in the beta-domain of MT1.     

Immunocytochemical localization of pigment-dispersing hormone (PDH) and its coexistence with FMRFamide-immunoreactive material in the eyestalks of the decapod crustaceans Carcinus maenas and Orconectes limosus

Summary By use of a new antiserum, raised against synthetic pigment-dispersing hormone (PDH) from Uca pugilator, immunoreactive structures were studied at the light-microscopic level in the eyestalk ganglia of Carcinus maenas and Orconectes limosus. PDH-reactivity was mainly found in two types of neurons that were located between the medulla interna (MI) and the medulla terminalis (MT) in both species. Several additional perikarya were located in the distal part of the MI in O. limosus. In C. maenas, two to three PDH-positive perikarya were found in the region of the X-organ (XO) in the MT. Processes from single and clustered cells could be traced into all medullae of the eyestalk. Axons from the immunoreactive perikarya running between MI and MT form a larger tract that traverses the MT. Fibers from this tract give rise to extensive arborizations and plexuses throughout the proximal MT. A plexus containing very fine fibers is located at the surface of the MT in a position distal to the XO-area of C. maenas only. The proximal plexus also receives PDH-positive fibers through the optic nerve. PDH-perikarya in the cerebral ganglion may also project into the more distal regions of the eyestalk. Distal projections of the perikarya between the MI and MT consist of several branches. Most of these are directed toward the MI and ME (medulla externa) wherein they form highly organized, layered plexuses. One branch was traced into the principal neurohemal organ, the sinus gland (SG). In the SG, the tract gives off arborizations and neurosecretory terminals. It then proceeds in a proximal direction out of the SG, adjacent to the MT. Its further course could not be elucidated. The lamina ganglionaris (LG) receives PDH-fibers from the ME and fine processes from small perikarya located in close association with the LG in the distal part of the first optic chiasma. The architecture of PDH-positive elements was similar in both C. maenas and O. limosus. The distribution of these structures suggests that PDH is not only a neurohormone but may, in addition, have a role as a neurotransmitter or modulator. Immunostaining of successive sections with an FMRF-amide antiserum revealed co-localization of FMRFamideand PDH-immunoreactivities in most, but not all PDH-containing perikarya and fibers. The axonal branch leading to the SG and the SG proper were devoid of FMRFamide immunoreactivity.     

Development ofDentalium following removal of D-quadrant blastomeres at successive cleavage stages

Summary Each primary micromere and macromere of the D-quadrant ofDentalium was deleted, through the mesentoblast stage, to investigate the way in which the polar lobe cytoplasm exerts its influence on development.-D and -1D embryos form an apical tuft but no posttrochal structures.-2D embryos form an apical tuft and a reduced posttrochal region without a shell. -3D and -4D are externally similar to control embryos. -1d embryos and -1c embryos have an apical tuft with a reduced number of cilia. Embryos in which both 1c and 1d are deleted lack the apical tuft.-2d embryos lack shell and most other posstrochal structures. -3d and-4d embryos appear externally equivalent to controls.The polar lobe cytoplasm exerts its influence sequentially, and as inIlyanassa the maximal effect is at the third quartet stage.     

Identification of microtubule binding sites in the Ncd tail domain

Nonclaret disjunctional (Ncd) is a minus end-directed, C-terminal motor protein that is required for spindle assembly and maintenance during meiosis and early mitosis in Drosophila oocytes and early embryos. Ncd has an ATP-independent MT binding site in the N-terminal tail domain, and an ATP-dependent MT binding site in the C-terminal motor domain. The ability of Ncd to cross-link MTs through the action of these binding sites may be important for Ncd function in vivo. To identify the region(s) responsible for ATP-independent MT interactions of Ncd, 12 cDNAs coding various regions of Ncd tail domain were expressed in E. coli as C-terminal fusions to thioredoxin (Trx). Ncd tail fusion proteins (TrxNT) were purified by ion exchange (S-Sepharose) and/or Talon metal affinity chromatography. Purified TrxNT and NT proteins were analyzed in microtubule (MT) cosedimentation and bundling assays to identify which tail proteins were able to bind and bundle MTs. Based on the results of these experiments, all TrxNT and NT proteins that showed MT binding activity also bundled MTs, and there are two ATP-independent MT interaction sites in the tail region: one within amino acids 83-100 that exhibits conformation-independent, high-affinity MT binding activity; and another within amino acids 115-187 that exhibits conformation-dependent, lower affinity MT binding activity. It is possible that both of these MT interacting sites combine in the native protein to form a single MT binding site that allows the Ncd tail to bind cargo MTs in vivo.     

Functional and structural evaluation of cysteine residues in the human arsenic (+3 oxidation state) methyltransferase (hAS3MT)

Arsenic (+3 oxidation state) methyltransferase (AS3MT) catalyzes the methylation of inorganic arsenic (iAs) and plays important role in the detoxication of this metalloid. There are fourteen cysteine residues in the human AS3MT (hAS3MT), among which twelve are absolutely conserved; Cys334 and Cys360 are unique; Cys368 and Cys369 are identified as a CysCys pair. The roles of several conserved cysteine residues in rat AS3MT and hAS3MT have been reported. Herein, the other conserved cysteine residues (Cys72, Cys271, Cys375) and the unique ones (Cys334, Cys360) were systematically replaced by serine using site-directed mutagenesis to study their functions. The mutants were investigated for enzymatic activity, kinetics, thermal stability and secondary structures. Present results indicate that C72S is completely inactive in methylation of iAs and has distinct changes in the secondary structures; Cys72 might form a critical intramolecular disulfide bond with Cys250; Cys271 and Cys375 do not affect the activity and structure of the hAS3MT. However, the mutations of Cys334 and Cys360 can decrease the enzymatic turnovers and change the conformation of the hAS3MT. The kinetic data show that Cys271, Cys334, Cys360 and Cys375 are not involved in the SAM binding. Additionally, all these cysteine residues except Cys375 affect the thermotropic properties of the hAS3MT.     

Two metal-binding peptides from the insect Orchesella cincta (Collembola) as a result of metallothionein cleavage

Metallothionein (MT) is an ubiquitous heavy metal-binding protein which has been identified in animals, plants, protists, fungi and bacteria. In insects, primary structures of MTs are known only for Drosophila and the collembolan, Orchesella cincta. The MT cDNA from O. cincta encodes a 77 amino acid protein with 19 cysteines. Isolations of the protein itself have demonstrated the presence of two smaller metal-binding peptides, whose amino acid sequences correspond to parts of the cDNA, and which apparently result from cleavage of the native protein. The present study was undertaken to complete the picture of cleavage sites within the MT protein by applying protein isolation techniques in combination with mass spectrometry and N-terminal sequence analysis. Further, recombinant expression allowed us to study the intrinsic stability of the MT and to perform in vitro cleavage studies. The results show that the MT from O. cincta is specifically cleaved at two sites, both after the amino acid sequence Thr-Gln (TQ). One of these sites is located in the N-terminal region and the other in the linker region between two putative metal-binding clusters. When expressed in Escherichia coli, the recombinant O. cincta MT can be isolated in an uncleaved form; however, this protein can be cleaved in vitro by the proteolytic activity of O. cincta. In combination with other studies, the results suggest that the length of the linker region is important for the stability of MT as a two domain metal-binding protein.     

Cytoskeleton of the mouse egg and embryo: reorganization of planar elements

Examination of detergent-extracted mouse eggs and embryos reveals the existence of two cytoskeletal networks. One network is the typical thin filament network observed in somatic cells while the other is composed of large planar elements. These latter cytoskeletal structures, with individual widths of 60.0 +/- 6.8 nm, alter their spatial organization in a developmental stage-specific manner. The planar elements are composed of filaments with a diameter of 10 nm aligned side-by-side with these filaments exhibiting a linear periodicity of 20.0 +/- 1.6 nm. A biochemical fraction containing components of the planar elements has been prepared from different stages of development and disappearance of prominent polypeptides from this fraction correlates with the altered spatial organization of the planar elements. Ultrastructure and biochemistry of cytoskeletal planar elements in eggs and embryos of the mouse are comparable with cytoskeletal sheets of Syrian hamster eggs and embryos, suggesting these cytoskeletal components may have a functional role in mammalian embryogenesis. Because such structures have not been identified in eggs or embryos of species other than mammals, their function may be unique to mammalian embryogenesis.     

Three-dimensional structural characterization of centrosomes from early Drosophila embryos

An understanding of the mechanism and structure of microtubule (MT)- nucleating sites within the pericentriolar material (PCM) of the centrosome has been elusive. This is partly due to the difficulty in obtaining large quantities of centrosomes for analysis, as well as to the problem of attaining interpretable structural data with conventional EM techniques. We describe a protocol for isolating a large quantity of functional centrosomes from early Drosophila embryos. Using automated electron tomography, we have begun a three-dimensional structural characterization of these intact centrosomes with and without regrown MTs. Reconstructions of the centrosomes to approximately 6-8 nm resolution revealed no large structures at the minus ends of MTs, suggesting that if MT-nucleating material physically contacts the MTs, it must conform closely to the shape of the minus end. While many MTs originate near the centrioles, MT minus ends were found throughout the PCM, and even close to its outer boundary. The MTs criss-crossed the PCM, suggesting that nucleating sites are oriented in many different directions. Reconstructions of centrosomes without MTs suggest that there is a reorganization of the PCM upon MT regrowth; moreover, ring-like structures that have a similar diameter as MTs are apparent in the PCM of centrosomes without MTs, and may be MT- nucleating sites.