Jorge Muschietti

Concerted regulation of nuclear and cytoplasmic activities of SR proteins by AKT.

Serine/arginine-rich (SR) proteins are important regulators of mRNA splicing. Several postsplicing activities have been described for a subset of shuttling SR proteins, including regulation of mRNA export and translation. Using the fibronectin gene to study the links between signal-transduction pathways and SR protein activity, we show that growth factors not only modify the alternative splicing pattern of the fibronectin gene but also alter translation of reporter messenger RNAs in an SR protein–dependent fashion, providing two coregulated levels of isoform-specific amplification. These effects are inhibited by specific small interfering RNAs against SR proteins and are mediated by the AKT kinase, which elicits opposite effects to those evoked by overexpressing SR protein kinases Clk and SRPK. These results show how SR protein activity is modified in response to extracellular stimulation, leading to a concerted regulation of splicing and translation.

A Cysteine-Rich Extracellular Protein, LAT52, Interacts with the Extracellular Domain of the Pollen Receptor Kinase LePRK2

Pollen germination and pollen tube growth are thought to require extracellular cues, but how these cues are perceived and transduced remains largely unknown. Pollen receptor kinases are plausible candidates for this role; they might bind extracellular ligands and thereby mediate cytoplasmic events required for pollen germination and pollen tube growth. To search for pollen-expressed ligands for pollen receptor kinases, we used the extracellular domains of three pollen-specific receptor kinases of tomato (LePRK1, LePRK2, and LePRK3) as baits in a yeast two-hybrid screen. We identified numerous secreted or plasma membrane–bound candidate ligands. One of these, the Cys-rich protein LAT52, was known to be essential during pollen hydration and pollen tube growth. We used in vivo coimmunoprecipitation to demonstrate that LAT52 was capable of forming a complex with LePRK2 in pollen and to show that the extracellular domain of LePRK2 was sufficient for the interaction. Soluble LAT52 can exist in differently sized forms, but only the larger form can interact with LePRK2. We propose that LAT52 might be a ligand for LePRK2.

Pollen tube localization implies a role in pollen-pistil interactions for the tomato receptor-like protein kinases LePRK1 and LePRK2.

We screened for pollen-specific kinase genes, which are potential signal transduction components of pollen–pistil interactions, and isolated two structurally related receptor-like kinases (RLKs) from tomato, LePRK1 and LePRK2. These kinases are similar to a pollen-expressed RLK from petunia, but they are expressed later during pollen development than is the petunia RLK. The abundance of LePRK2 increases when pollen germinates, but LePRK1 remains constant. Both LePRK1 and LePRK2 are localized to the plasma membrane/cell wall of growing pollen tubes. Both kinase domains have kinase activity when expressed in Escherichia coli. In phosphorylation assays with pollen membrane preparations, LePRK2, but not LePRK1, is phosphorylated, and the addition of tomato style, but not leaf, extracts to these membrane preparations results at least partially in specific dephosphorylation of LePRK2. Taken together, these results suggest that LePRK1 and LePRK2 play different roles in postpollination events and that at least LePRK2 may mediate some pistil response.

Bicarbonate dependence of cAMP accumulation induced by phorbol esters in hamster spermatozoa

Phorbol esters stimulate cyclic adenosine 3,5-monophosphate (cAMP) accumulation in hamster spermatozoa under conditions for in vitro capacitation. The 20-50-fold elevation of cAMP levels induced by 1 microM phorbol 12-myristate 13-acetate (PMA) in spermatozoa depends on the presence of sodium bicarbonate in the medium (ED50: 15 mM) and it is independent of extracellular pH. Sodium bicarbonate stimulates adenylate cyclase activity in membrane preparations by 4-fold (ED50: 40 mM). After solubilization, the bicarbonate-sensitive moiety elutes as a single peak of 55 kDa in a gel filtration column. Blockers of bicarbonate chloride antiporters diisothiocyanate stilbene 2,2-disulfonic acid (DIDS) or acetamido 4-isothiocyanate stilbene 2,2-disulfonic acid (SITS) inhibit the bicarbonate dependent PMA effect on cAMP in living spermatozoa (ED50: 100 microM). Maximal (85%) inhibition in cAMP accumulation is observed at 1 mM. Motility is inhibited only at high concentrations of the blockers. Pretreatment of living cells with 1 mM DIDS does not affect membrane adenylate cyclase activity which remains responsive to bicarbonate. These results suggest that controlled transport of bicarbonate through the sperm plasma membrane could be associated to the regulation of cAMP synthesis.

The Pollen Receptor Kinase LePRK2 Mediates Growth-Promoting Signals and Positively Regulates Pollen Germination and Tube Growth

In flowering plants, the process of pollen germination and tube growth is required for successful fertilization. A pollen receptor kinase from tomato (Solanum lycopersicum), LePRK2, has been implicated in signaling during pollen germination and tube growth as well as in mediating pollen (tube)-pistil communication. Here we show that reduced expression of LePRK2 affects four aspects of pollen germination and tube growth. First, the percentage of pollen that germinates is reduced, and the time window for competence to germinate is also shorter. Second, the pollen tube growth rate is reduced both in vitro and in the pistil. Third, tip-localized superoxide production by pollen tubes cannot be increased by exogenous calcium ions. Fourth, pollen tubes have defects in responses to style extract component (STIL), an extracellular growth-promoting signal from the pistil. Pollen tubes transiently overexpressing LePRK2-fluorescent protein fusions had slightly wider tips, whereas pollen tubes coexpressing LePRK2 and its cytoplasmic partner protein KPP (a Rop-GEF) had much wider tips. Together these results show that LePRK2 positively regulates pollen germination and tube growth and is involved in transducing responses to extracellular growth-promoting signals.

Prediction of Aquaporin Function by Integrating Evolutionary and Functional Analyses

Aquaporins (AQPs) are a family of channel proteins, which transport water and/or small solutes across cell membranes. AQPs are present in Bacteria, Eukarya, and Archaea. The classical AQP evolution paradigm explains the inconsistent phylogenetic trees by multiple transfer events and emphasizes that the assignment of orthologous AQPs is not possible, making it difficult to integrate functional information. Recently, a novel phylogenetic framework of eukaryotic AQP evolution showed congruence between eukaryotic AQPs and organismal trees identifying 32 orthologous clusters in plants and animals (Soto et al. Gene 503:165–176, 2012). In this article, we discuss in depth the methodological strength, the ability to predict functionality and the AQP community perception about the different paradigms of AQP evolution. Moreover, we show an updated review of AQPs transport functions in association with phylogenetic analyses. Finally, we discuss the possible effect of AQP data integration in the understanding of water and solute transport in eukaryotic cells.

Hormonal networks involved in apical hook development in darkness and their response to light

In darkness, the dicot seedlings produce an apical hook as result of differential cell division and extension at opposite sides of the hypocotyl. This hook protects the apical meristem from mechanical damage during seedling emergence from the soil. In darkness, gibberellins act via the DELLA-PIF (PHYTOCHROME INTERACTING FACTORs) pathway, and ethylene acts via the EIN3/EIL1 (ETHYLENE INSENSITIVE 3/EIN3 like 1)-HLS1 (HOOKLESS 1) pathway to control the asymmetric accumulation of auxin required for apical hook formation and maintenance. These core pathways form a network with multiple points of connection. Light perception by phytochromes and cryptochromes reduces the activity of PIFs and (COP1) CONSTITUTIVE PHOTOMORPHOGENIC 1—both required for hook formation in darkness—, lowers the levels of gibberellins, and triggers hook opening as a component of the switch between heterotrophic and photoautotrophic development. Apical hook opening is thus a suitable model to study the convergence of endogenous and exogenous signals on the control of cell division and cell growth.

Overexpression of the Tomato Pollen Receptor Kinase LePRK1 Rewires Pollen Tube Growth to a Blebbing Mode

Pollen tubes are tubular structures that extend by tip growth. Upon overexpression of merely a single endogenous molecule, LePRK1, or a portion of it lacking its extracellular domain, pollen tube cells switched to an alternative mechanism for extension of the leading edge that is analogous to the blebbing growth mode reported for Dictyostelium and for Drosophila stem cells. The tubular growth of a pollen tube cell is crucial for the sexual reproduction of flowering plants. LePRK1 is a pollen-specific and plasma membrane–localized receptor-like kinase from tomato (Solanum lycopersicum). LePRK1 interacts with another receptor, LePRK2, and with KINASE PARTNER PROTEIN (KPP), a Rop guanine nucleotide exchange factor. Here, we show that pollen tubes overexpressing LePRK1 or a truncated LePRK1 lacking its extracellular domain (LePRK1ΔECD) have enlarged tips but also extend their leading edges by producing “blebs.” Coexpression of LePRK1 and tomato PLIM2a, an actin bundling protein that interacts with KPP in a Ca2+-responsive manner, suppressed these LePRK1 overexpression phenotypes, whereas pollen tubes coexpressing KPP, LePRK1, and PLIM2a resumed the blebbing growth mode. We conclude that overexpression of LePRK1 or LePRK1ΔECD rewires pollen tube growth to a blebbing mode, through KPP- and PLIM2a-mediated bundling of actin filaments from tip plasma membranes. Arabidopsis thaliana pollen tubes expressing LePRK1ΔECD also grew by blebbing. Our results exposed a hidden capability of the pollen tube cell: upon overexpression of a single membrane-localized molecule, LePRK1 or LePRK1ΔECD, it can switch to an alternative mechanism for extension of the leading edge that is analogous to the blebbing growth mode reported for Dictyostelium and for Drosophila melanogaster stem cells.

Comparisons of diverse plant species reveal that only grasses show drastically reduced levels of ubiquitin monomer in mature pollen

Ubiquitin is a ubiquitous protein involved in targeting proteins for degradation. Maize pollen was previously reported (Callis and Bedinger 1994) to show extremely low levels of ubiquitin monomer, and developmental significance was attributed to this surprising feature of maize pollen. However, we had previously shown (Muschietti et al. 1994) that tomato pollen had high levels of ubiquitin monomer. Here we show that pollen from most plant families has high levels of ubiquitin monomer. Most grasses tested show reduced levels of ubiquitin monomer, but some maize inbred lines have higher levels of ubiquitin monomer than other inbreds. There was no correlation between the level of ubiquitin monomer and either the monocotyledonous or tri-cellular condition of grass pollen or the dehydrated condition of mature pollen. Since many aspects of pollen development (i.e., wall formation, microspore mitosis, synthesis and storage of mRNAs and proteins, carbohydrates and lipids, dehydration at maturity) are stereotypical among all plant families, the reduced level of ubiquitin monomer in pollen of many grasses cannot be crucial for any feature of normal pollen development.

Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi.

A soluble nucleoside diphosphate kinase (NDP kinase) was purified and characterized in epimastigote forms of Trypanosoma cruzi. The enzyme was purified by affinity chromatography on Blue-agarose and Q-Sepharose columns and by FPLC on a Superose 12 column. A membrane-associated NDP kinase was identified which accounts for 30% of total enzymatic activity. Western blot analysis of the soluble NDP kinase revealed a 16.5-kDa monomer recognized by polyclonal antibodies to NDP kinase from Dictyostelium discoideum, Candida albicans or human. Most of the T. cruzi NDP kinase is found in the cell as a hexamer composed of 16.5-kDa monomers. The Km values of the enzyme for ATP, GDP and dTDP were 0.2 +/- 0.008 mM, 0.125 +/- 0.012 mM and 0.4 +/- 0.009 mM, respectively. The parasite enzyme was stable, remained active at 65 degrees C and was found to tolerate up to 2.5 M urea. The 16.5-kDa subunit was phosphorylated with [gamma-32P]ATP or thiophosphorylated with [35S]GTP gamma S. The incubation of the 32P-labelled phosphoenzyme with unlabelled nucleoside 5-diphosphates resulted in the formation of 32P-labelled nucleoside 5-triphosphates without strict base specificity, indicating that the reaction mechanism of the T. cruzi enzyme is the same as reported for other NDP kinases. When the phosphoenzyme was incubated with a mixture of nucleoside 5-diphosphates, GTP was preferentially formed.