Khaled Machaca

RNA interference is an antiviral defence mechanism in Caenorhabditis elegans

RNA interference (RNAi) is an evolutionarily conserved sequence-specific post-transcriptional gene silencing mechanism that is well defined genetically in Caenorhabditis elegans. RNAi has been postulated to function as an adaptive antiviral immune mechanism in the worm, but there is no experimental evidence for this. Part of the limitation is that there are no known natural viral pathogens of C. elegans. Here we describe an infection model in C. elegans using the mammalian pathogen vesicular stomatitis virus (VSV) to study the role of RNAi in antiviral immunity. VSV infection is potentiated in cells derived from RNAi-defective worm mutants (rde-1; rde-4), leading to the production of infectious progeny virus, and is inhibited in mutants with an enhanced RNAi response (rrf-3; eri-1). Because the RNAi response occurs in the absence of exogenously added VSV small interfering RNAs, these results show that RNAi is activated during VSV infection and that RNAi is a genuine antiviral immune defence mechanism in the worm.

Orai1 internalization and STIM1 clustering inhibition modulate SOCE inactivation during meiosis

Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ influx pathway activated in response to depletion of intracellular Ca2+ stores. SOCE is a primary modulator of intracellular Ca2+ dynamics, which specify cellular responses. Interestingly, SOCE inactivates during M phase but the mechanisms involved remain unclear. SOCE is mediated by clustering of the ER Ca2+ sensor STIM1 in response to Ca2+ store depletion, leading to gating of the plasma membrane SOCE channel Orai1. Here we show that SOCE inactivation in meiosis is the result of internalization of Orai1 into an intracellular vesicular compartment and to the inability of STIM1 to cluster in response to store depletion. At rest, Orai1 continuously recycles between the cell membrane and an endosomal compartment. We further show that STIM1–STIM1 interactions are inhibited during meiosis, which appears to mediate the inability of STIM1 to form puncta following store depletion. In contrast, STIM1–Orai1 interactions remain functional during meiosis. Combined, the removal of Orai1 from the cell membrane and STIM1 clustering inhibition effectively uncouple store depletion from SOCE activation in meiosis. Although STIM1 is phosphorylated during meiosis, phosphomimetic and alanine substitution mutations do not modulate STIM1 clustering, arguing that phosphorylation does not mediate STIM1 clustering inhibition during meiosis.

Constitutive recycling of the store-operated Ca2+ channel Orai1 and its internalization during meiosis.

Orai1 trafficking between the cell membrane and endosomes during meiosis requires caveolin and is important for tuning calcium signaling during oocyte maturation.

Induction of maturation-promoting factor during Xenopus oocyte maturation uncouples Ca2+ store depletion from store-operated Ca2+ entry

Department of Physiology and Biophysics, University of Arkansas Medical Science, Little Rock, AR 72205 During oocyte maturation, eggs acquire the ability to generate specialized Ca2+ signals in response to sperm entry. Such Ca2+ signals are crucial for egg activation and the initiation of embryonic development. We examined the regulation during Xenopus oocyte maturation of store-operated Ca2+ entry (SOCE), an important Ca2+ influx pathway in oocytes and other nonexcitable cells. We have previously shown that SOCE inactivates during Xenopus oocyte meiosis. SOCE inactivation may be important in preventing premature egg activation. In this study, we investigated the correlation between SOCE inactivation and the Mos–mitogen-activated protein kinase (MAPK)–maturation-promoting factor (MPF) kinase cascade, which drives Xenopus oocyte maturation. SOCE inactivation at germinal vesicle breakdown coincides with an increase in the levels of MAPK and MPF. By differentially inducing Mos, MAPK, and MPF, we demonstrate that the activation of MPF is necessary for SOCE inactivation during oocyte maturation. In contrast, sustained high levels of Mos kinase and the MAPK cascade have no effect on SOCE activation. We further show that preactivated SOCE is not inactivated by MPF, suggesting that MPF does not block Ca2+ influx through SOCE channels, but rather inhibits coupling between store depletion and SOCE activation.

Ca2+ signaling differentiation during oocyte maturation

Oocyte maturation is an essential cellular differentiation pathway that prepares the egg for activation at fertilization leading to the initiation of embryogenesis. An integral attribute of oocyte maturation is the remodeling of Ca2+ signaling pathways endowing the egg with the capacity to produce a specialized Ca2+ transient at fertilization that is necessary and sufficient for egg activation. Consequently, mechanistic elucidation of Ca2+ signaling differentiation during oocyte maturation is fundamental to our understanding of egg activation, and offers a glimpse into Ca2+ signaling regulation during the cell cycle. J. Cell. Physiol. 213: 331–340, 2007.

Understanding fertilization through intracytoplasmic sperm injection (ICSI)

Since the establishment of in vitro fertilization, it became evident that almost half of the couples failed to achieve fertilization and this phenomenon was attributed to a male gamete dysfunction. The adoption of assisted fertilization techniques particularly ICSI has been able to alleviate male factor infertility by granting the consistent ability of a viable spermatozoon to activate an oocyte. Single sperm injection, by pinpointing the beginning of fertilization, has been an invaluable tool in clarifying the different aspects of early fertilization and syngamy. However, even with ICSI some couples fail to fertilize due to ooplasmic dysmaturity in relation to the achieved nuclear maturation marked by the extrusion of the first polar body. More uncommon are cases where the spermatozoa partially or completely lack the specific oocyte activating factor. In this work, we review the most relevant aspects of fertilization and its failure through assisted reproductive technologies. Attempts at diagnosing and treating clinical fertilization failure are described.

Ca2+ signaling, genes and the cell cycle

Changes in the concentration and spatial distribution of Ca(2+) ions in the cytoplasm constitute a ubiquitous intracellular signaling module in cellular physiology. With the advent of Ca(2+) dyes that allow direct visualization of Ca(2+) transients, combined with powerful experimental tools such as electrophysiological recordings, intracellular Ca(2+) transients have been implicated in practically every aspect of cellular physiology, including cellular proliferation. Ca(2+) signals are associated with different phases of the cell cycle and interfering with Ca(2+) signaling or downstream pathways often disrupts progression of the cell cycle. Although there exists a dependence between Ca(2+) signals and the cell cycle the mechanisms involved are not well defined and given the cross-talk between Ca(2+) and other signaling modules, it is difficult to assess the exact role of Ca(2+) signals in cell cycle progression. Two exceptions however, include fertilization and T-cell activation, where well-defined roles for Ca(2+) signals in mediating progression through specific stages of the cell cycle have been clearly established. In the case of T-cell activation Ca(2+) regulates entry into the cell cycle through the induction of gene transcription.

Ca2+cyt negatively regulates the initiation of oocyte maturation

Ca2+ is a ubiquitous intracellular messenger that is important for cell cycle progression. Genetic and biochemical evidence support a role for Ca2+ in mitosis. In contrast, there has been a long-standing debate as to whether Ca2+ signals are required for oocyte meiosis. Here, we show that cytoplasmic Ca2+ (Ca2+ cyt) plays a dual role during Xenopus oocyte maturation. Ca2+ signals are dispensable for meiosis entry (germinal vesicle breakdown and chromosome condensation), but are required for the completion of meiosis I. Interestingly, in the absence of Ca2+ cyt signals oocytes enter meiosis more rapidly due to faster activation of the MAPK-maturation promoting factor (MPF) kinase cascade. This Ca2+-dependent negative regulation of the cell cycle machinery (MAPK-MPF cascade) is due to Ca2+ cyt acting downstream of protein kinase A but upstream of Mos (a MAPK kinase kinase). Therefore, high Ca2+ cyt delays meiosis entry by negatively regulating the initiation of the MAPK-MPF cascade. These results show that Ca2+ modulates both the cell cycle machinery and nuclear maturation during meiosis.

Intramolecular shielding maintains the ER Ca2+ sensor STIM1 in an inactive conformation

Summary Store-operated calcium entry (SOCE) represents a major calcium influx pathway in non-excitable cells and is central to many physiological processes such as T cell activation and mast cell degranulation. SOCE is activated through intricate coordination between the Ca2+ sensor on the ER membrane (stromal interaction molecule 1, STIM1) and the plasma membrane channel Orai1. When Ca2+ stores are depleted, STIM1 oligomerizes and physically interacts with Orai1 through its SOAR/CAD domain, resulting in Orai1 gating and Ca2+ influx. Here, we describe novel inter- and intramolecular FRET sensors in the context of the full-length membrane-anchored STIM1, and show that STIM1 undergoes a conformational change in response to store depletion to adopt a stretched ‘open’ conformation that exposes SOAR/CAD and allows it to interact with Orai1. Mutational analyses reveal that electrostatic interactions between the predicted first and third coiled-coil domains of STIM1 are not involved in maintaining the ‘closed’ inactive conformation. In addition, the results argue that an amphipathic &agr;-helix between residues 317 and 336 in the so-called inhibitory domain is important to maintain STIM1 in a closed conformation at rest. Indeed, mutations that alter the amphipathic properties of this helix result in a STIM1 variant that is unable to respond to store depletion in terms of forming puncta, translocation to the cortical ER or activating Orai1.

Copper chelation selectively kills colon cancer cells through redox cycling and generation of reactive oxygen species

BackgroundMetals including iron, copper and zinc are essential for physiological processes yet can be toxic at high concentrations. However the role of these metals in the progression of cancer is not well defined. Here we study the anti-tumor activity of the metal chelator, TPEN, and define its mechanism of action.MethodsMultiple approaches were employed, including cell viability, cell cycle analysis, multiple measurements of apoptosis, and mitochondrial function. In addition we measured cellular metal contents and employed EPR to record redox cycling of TPEN–metal complexes. Mouse xenografts were also performed to test the efficacy of TPEN in vivo.ResultsWe show that metal chelation using TPEN (5μM) selectively induces cell death in HCT116 colon cancer cells without affecting the viability of non-cancerous colon or intestinal cells. Cell death was associated with increased levels of reactive oxygen species (ROS) and was inhibited by antioxidants and by prior chelation of copper. Interestingly, HCT116 cells accumulate copper to 7-folds higher levels than normal colon cells, and the TPEN-copper complex engages in redox cycling to generate hydroxyl radicals. Consistently, TPEN exhibits robust anti-tumor activity in vivo in colon cancer mouse xenografts.ConclusionOur data show that TPEN induces cell death by chelating copper to produce TPEN-copper complexes that engage in redox cycling to selectively eliminate colon cancer cells.