Evidence for involvement of metalloendoproteases in a step in sea urchin gamete fusion.

Membrane fusion events are required in three steps in sea urchin fertilization: the acrosome reaction in sperm, fusion of the plasma membrane of acrosome-reacted sperm with the plasma membrane of the egg, and exocytosis of the contents of the egg cortical granules. We recently reported the involvement of a Zn2+-dependent metalloendoprotease in the acrosome reaction (Farach, H. C., D. I. Mundy, W. J. Strittmatter, and W. J. Lennarz. 1987. J. Biol. Chem. 262:5483-5487). In the current study, we investigated the possible involvement of metalloendoproteases in the two other fusion events of fertilization. The use of inhibitors of metalloendoproteases provided evidence that at least one of the fusion events subsequent to the acrosome reaction requires such enzymes. These inhibitors did not block the binding of sperm to egg or the process of cortical granule exocytosis. However, sperm-egg fusion, assayed by the ability of the bound sperm to establish cytoplasmic continuity with the egg, was inhibited by metalloendoprotease substrate. Thus, in addition to the acrosome reaction, an event in the gamete fusion process requires a metalloendoprotease.

Biosynthesis and secretion of the hatching enzyme during sea urchin embryogenesis.

The hatching enzyme secreted by the blastula stage sea urchin embryo proteolyzes the fertilization envelope, thereby allowing the embryo to hatch. Using an assay that measures fertilization envelope degradation, we have purified the hatching enzyme by ion-exchange and affinity chromatography from the hatching medium of Strongylocentrotus purpuratus embryos. The hatching enzyme was found to be a 33-kDa metalloprotease that exhibited a substrate preference for only a minor subset of proteins in the fertilization envelope. Secretion of the hatching enzyme by blastula stage embryos occurred during the 2-h period prior to hatching. The hatching enzyme was initially secreted as a 57-kDa protein, but during purification it was converted to the 33-kDa form. Biosynthesis of the hatching enzyme began at the late morula/early blastula stage up to 6 h before secretion. Experiments using the ionophore monensin suggest that the lag between synthesis and secretion of the hatching enzyme by the blastula stage embryos may be a result of a slow constitutive secretory process.

TOUSLED participates in apical tissue formation during gynoecium development in Arabidopsis.

Mutations at the TOUSLED (TSL) protein kinase locus in Arabidopsis cause reduced differentiation of apical gynoecial tissues and eliminate the fusion of the style and septum. TSL expression becomes confined to the developing style by stage 13, where it may promote expansion of tissues. Double mutant analysis suggests that ETTIN interacts with TSL, possibly by restricting TSL expression to apical regions. TSL, LEUNIG, and PERIANTHIA appear to participate in pathways of redundant function during the development of specific gynoecial tissues. TSL and LEUNIG most likely function in similar pathways during ovule development. TSL acts independently of the function of the organ identity genes AGAMOUS and APETALA2, and it is required for the formation of specific tissues in ectopic carpels. Mutations in TSL, ETTIN, PERIANTHIA, and LEUNIG all affect floral organ number as well as gynoecium morphology. Their respective wild-type loci must therefore play important roles in early floral meristem development during initiation of organ primordia in addition to their functions during regional differentiation within developing gynoecial primordia.

Inhibitors of metalloendoproteases block spiculogenesis in sea urchin primary mesenchyme cells.

Metalloendoproteases have been implicated in a variety of fusion processes including plasma membrane fusion and exocytosis. As a prerequisite to skeleton formation in the sea urchin embryo, primary mesenchyme cells undergo fusion via filopodia to form syncytia. The spicule is formed within the syncytial cable by matrix and mineral deposition. To investigate the potential involvement of a metalloendoprotease in spiculogenesis, the effect of inhibitors of this enzyme on skeleton formation was studied. Experiments with primary mesenchyme cells in vitro and in normal embryos revealed that skeleton formation was blocked by these inhibitors. These findings implicate a metalloendoprotease in spiculogenesis; such an enzyme has been demonstrated in homogenates of primary mesenchyme cells. The most likely site of action of the metalloendoprotease is at the cell membrane fusion stage and/or at subsequent events requiring membrane fusion.

Isolation and characterization of interferon-producing reticuloendothelial cells from mouse epidermis.

A homogeneous population of trypsin-resistant epidermal cells has been isolated from newborn ICR mice. These cells are characterized by adherence, receptors for Fc-IgG, ATPase activity, phagocytosis of latex particles and opsonized sheep erythrocytes, and secretion of lysozyme and interferon. The production of interferon by these cells suggests that they may be important in protection against viral infections of the skin as well as in regulation of immune responses. The ultrastructure of these trypsin-resistant epidermal cells shows striking similarity to that of reticuloendothelial cells.

TOUSLED is a nuclear serine/threonine protein kinase that requires a coiled-coil region for oligomerization and catalytic activity.

The TOUSLED (TSL) gene is essential for the proper morphogenesis of leaves and flowers in Arabidopsis thaliana. Protein sequence analysis predicts TSL is composed of a carboxyl-terminal protein kinase catalytic domain and a large amino-terminal regulatory domain. TSL fusion proteins, expressed in and purified from yeast, were used to demonstrate TSL protein kinase activity in vitro. TSL trans-autophosphorylates on serine and threonine residues, and phosphorylates exogenous substrates. Using the yeast two-hybrid system, TSL was found to oligomerize via its NH2-terminal domain. A deletion series indicates that a region containing two alpha-helical segments predicted to participate in a coiled-coil structure is essential for oligomerization. TSL localizes to the nucleus in plant cells through an essential NH2-terminal nuclear localization signal; however, this signal is not necessary for protein kinase activity. Finally, deletion mutants demonstrate a strict correlation between catalytic activity and the ability to oligomerize, arguing that activation of the protein kinase requires interaction between TSL molecules.

ETTIN patterns the Arabidopsis floral meristem and reproductive organs.

ettin (ett) mutations have pleiotropic effects on Arabidopsis flower development, causing increases in perianth organ number, decreases in stamen number and anther formation, and apical-basal patterning defects in the gynoecium. The ETTIN gene was cloned and encodes a protein with homology to DNA binding proteins which bind to auxin response elements. ETT transcript is expressed throughout stage 1 floral meristems and subsequently resolves to a complex pattern within petal, stamen and carpel primordia. The data suggest that ETT functions to impart regional identity in floral meristems that affects perianth organ number spacing, stamen formation, and regional differentiation in stamens and the gynoecium. During stage 5, ETT expression appears in a ring at the top of the floral meristem before morphological appearance of the gynoecium, consistent with the proposal that ETT is involved in prepatterning apical and basal boundaries in the gynoecium primordium. Double mutant analyses and expression studies show that although ETT transcriptional activation occurs independently of the meristem and organ identity genes LEAFY, APETELA1, APETELA2 and AGAMOUS, the functioning of these genes is necessary for ETT activity. Double mutant analyses also demonstrate that ETT functions independently of the 'b' class genes APETELA3 and PISTILLATA. Lastly, double mutant analyses suggest that ETT control of floral organ number acts independently of CLAVATA loci and redundantly with PERIANTHIA.

The Tousled gene in A. thaliana encodes a protein kinase homolog that is required for leaf and flower development.

Mutation at the TOUSLED locus of A. thaliana results in a complex phenotype, the most dramatic aspect of which being the abnormal flowers produced in mutant plants. tsl flowers show a random loss of floral organs, and organ development is impaired. The TSL gene appears to be required in the floral meristem for correct initiation of floral organ primordia and for proper development of organ primordia. Loss of TSL function also affects flowering time and leaf morphology. Using a mutation derived by T-DNA insertion mutagenesis, we have cloned the TSL gene and found that it encodes a protein kinase homolog with a novel N-terminal domain. This protein kinase gene identifies a novel signaling/regulatory pathway used during development in Arabidopsis.

A geminivirus replication protein interacts with the retinoblastoma protein through a novel domain to determine symptoms and tissue specificity of infection in plants.

Geminiviruses replicate in nuclei of mature plant cells after inducing the accumulation of host DNA replication machinery. Earlier studies showed that the viral replication factor, AL1, is sufficient for host induction and interacts with the cell cycle regulator, retinoblastoma (pRb). Unlike other DNA virus proteins, AL1 does not contain the pRb binding consensus, LXCXE, and interacts with plant pRb homo logues (pRBR) through a novel amino acid sequence. We mapped the pRBR binding domain of AL1 between amino acids 101 and 180 and identified two mutants that are differentially impacted for AL1-pRBR interactions. Plants infected with the E-N140 mutant, which is wild-type for pRBR binding, developed wild-type symptoms and accumulated viral DNA and AL1 protein in epidermal, mesophyll and vascular cells of mature leaves. Plants inoculated with the KEE146 mutant, which retains 16% pRBR binding activity, only developed chlorosis along the veins, and viral DNA, AL1 protein and the host DNA synthesis factor, proliferating cell nuclear antigen, were localized to vascular tissue. These results established the importance of AL1-pRBR interactions during geminivirus infection of plants.