Quantitative 3-D imaging of eukaryotic cells using soft X-ray tomography.

Imaging has long been one of the principal techniques used in biological and biomedical research. Indeed, the field of cell biology grew out of the first electron microscopy images of organelles in a cell. Since this landmark event, much work has been carried out to image and classify the organelles in eukaryotic cells using electron microscopy. Fluorescently labeled organelles can now be tracked in live cells, and recently, powerful light microscope techniques have pushed the limit of optical resolution to image single molecules. In this paper, we describe the use of soft X-ray tomography, a new tool for quantitative imaging of organelle structure and distribution in whole, fully hydrated eukaryotic Schizosaccharomyces pombe cells. In addition to imaging intact cells, soft X-ray tomography has the advantage of not requiring the use of any staining or fixation protocols--cells are simply transferred from their growth environment to a sample holder and immediately cryofixed. In this way the cells can be imaged in a near native state. Soft X-ray tomography is also capable of imaging relatively large numbers of cells in a short period of time, and is therefore a technique that has the potential to produce information on organelle morphology from statistically significant numbers of cells.

Tumorhead distribution to cytoplasmic membrane of neural plate cells is positively regulated by Xenopus p21-activated kinase 1 (X-PAK1).

Tumorhead (TH) regulates neural plate cell proliferation during Xenopus early development, and gain or loss of function prevents neural differentiation. TH shuttles between the nuclear and cytoplasmic/cortical cell compartments in embryonic cells. In this study, we show that subcellular distribution of TH is important for its functions. Targeting TH to the cell cortex/membrane potentiates a TH gain of function phenotype and results in neural plate expansion and inhibition of neuronal differentiation. We have found that TH subcellular localization is regulated, and that its shuttling between the nucleus and the cell cortex/cytoplasm is controlled by the catalytic activity of p21-activated kinase, X-PAK1. The phenotypes of embryos that lack, or have excess, X-PAK1 activity mimic the phenotypes induced by loss or gain of TH functions, respectively. We provide evidence that X-PAK1 is an upstream regulator of TH and discuss potential functions of TH at the cell cortex/cytoplasmic membrane and in the nucleus.

X-ray tomography of Schizosaccharomyces pombe.

The genetic tractability of the unicellular yeast Schizosaccharomyces pombe has resulted in it becoming an important model organism for the study of many eukaryotic cellular processes, in particular cell division. Over the past few years much progress has been made toward understanding the mechanisms that regulate eukaryotic cell division and the cellular changes that occur-for example, the formation of the cytokinetic contractile ring. However, a full understanding requires both identification of the proteins involved and correlation of this information with images showing the location of molecules in context of the cell architecture. Electron microscopic analyses have revealed exquisite ultrastructural images of cell structure, but this technique typically requires extensive processing-procedures that are labor intensive and time consuming. Imaging techniques that can more rapidly obtain better resolution than light microscopy are needed to advance the use of this model system for precise molecular localization analyses. In this manuscript, we examined S. pombe using soft X-ray tomography, an imaging technique that generates three-dimensional (3-D) images of intact hydrated cells at better than 50 nm isotropic resolution. This technique uses X-rays in the "water window," where organic material absorbs approximately an order of magnitude more strongly than water, producing high-contrast images of cellular structures. As cells are examined in the absence of any chemical fixatives, stains, or contrast enhancement reagents, the images reflect cellular structures in the near-native state. We conducted preliminary soft X-ray imaging of S. pombe cells before and during cell division that revealed subcellular organelles, the actomyosin ring, and the septum of dividing cells. These images reveal tantalizing details of the cytokinesis process and are the first steps in our goal of generating a portfolio of tomographic images that map the location of labeled molecules into high-resolution 3-D reconstructions of the cell.

Disruption of the dynamic sub-cellular localization of the Xenopus tumorhead protein causes embryonic lethality at the early gastrula transition.

The Xenopus laevis tumorhead (TH) protein, a positive regulator of cell proliferation during embryogenesis, shuttles from the cell periphery into the nucleus during embryogenesis. In these studies, we performed a detailed analysis of TH's subcellular localization pattern to characterize its dynamic behavior. We found that TH exhibits distinct patterns of localization in different germ layers. At the blastula stage, TH is present in the apical cell periphery of prospective mesodermal and ectodermal cells. At the gastrula stage, TH is distributed throughout the entire cytoplasm of prospective mesodermal and ectodermal cells, whereas it shows nuclear localization in presumptive endodermal cells. TH moves into the nucleus of mesodermal and ectodermal cells during the neurula and early tailbud stages. To understand if TH is regulated by changes in its subcellular localization, we used a TH mutant containing signals for farnesylation and palmitoylation to tether the protein to the plasma membrane. Ubiquitous overexpression of this mutant causes embryonic lethality at the early gastrula transition. Further examination using TUNEL assays indicated that wild-type TH overexpression induces apoptosis during gastrulation, and that this effect is exacerbated by the overexpression of the membrane-bound TH mutant. Taken together, our results suggest that changes in the sub-cellular localization of the TH protein are important for its function because blocking the nuclear translocation of overexpressed TH increases apoptosis and causes embryos to die. Our data also suggest that TH plays a role outside the nucleus when it is present at the cell periphery.

The maternally localized RNA fatvg is required for cortical rotation and germ cell formation.

Fatvg is a localized maternal transcript that translocates to the vegetal cortex of Xenopus laevis oocytes through both the METRO and Late RNA localization pathways. It is a member of a gene family that functions in vesicular trafficking. Depletion of the maternal store of fatvg mRNA results in a dual phenotype in which embryos are ventralized and also lack primordial germ cells. This complex fatvg loss of function phenotype is the result of stabilization of the dorsalizing factor beta-catenin at the vegetal pole and the inability of the germ cell determinants to move to their proper locations. This is coincident with the inhibition of cortical rotation and the abnormal aggregation of the germ plasm. Fatvg protein is located at the periphery of vesicles in the oocyte and embryo, supporting its proposed role in vesicular trafficking in the embryo. These results point to a common fundamental mechanism that is regulated by fatvg through which germ cell determinants and dorsalizing factors segregate during early development.

Hermes RNA-binding protein targets RNAs-encoding proteins involved in meiotic maturation, early cleavage, and germline development.

The early development of metazoans is mainly regulated by differential translation and localization of maternal mRNAs in the embryo. In general, these processes are orchestrated by RNA-binding proteins interacting with specific sequence motifs in the 3'-untranslated region (UTR) of their target RNAs. Hermes is an RNA-binding protein, which contains a single RNA recognition motif (RRM) and is found in various vertebrate species from fish to human. In Xenopus laevis, Hermes mRNA and protein are localized in the vegetal region of oocytes. A subpopulation of Hermes protein is concentrated in a specific structure in the vegetal cortex, called the germ plasm (believed to contain determinants of the germ cell fate) where Hermes protein co-localizes with Xcat2 and RINGO/Spy mRNAs. The level of total Hermes protein decreases during maturation. The precocious depletion of Hermes protein by injection of Hermes antisense morpholino oligonucleotide (HE-MO) accelerates the process of maturation and results in cleavage defects in vegetal blastomeres of the embryo. It is known that several maternal mRNAs including RINGO/Spy and Mos are regulated at the translational level during meiotic maturation and early cleavage in Xenopus. The ectopic expression of RINGO/Spy or Mos causes resumption of meiotic maturation and cleavage arrests, which resemble the loss of Hermes phenotypes. We found that the injection of HE-MO enhances the acceleration of maturation caused by the injection of RINGO/Spy mRNA, and that Hermes protein is present as mRNP complex containing RINGO/Spy, Mos, and Xcat2 mRNAs in vivo. We propose that as an RNA-binding protein, Hermes may be involved in maturation, cleavage events at the vegetal pole and germ cell development by negatively regulating the expression of RINGO/Spy, Mos, and Xcat2 mRNAs.

Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins.

The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (a-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.

Identification of TH-B, a new oligomeric variant of the Xenopus morphogenetic factor, tumorhead.

The Xenopus laevis gene tumorhead (TH) is a regulator of cell proliferation of the ectodermal germ layer during embryonic development. TH overexpression results in increased cell proliferation within the developing ectoderm, causing an expansion of the neural plate. Conversely, loss of TH function results in inhibition of proliferation of ectodermal cells. Embryos with altered levels of TH protein are unable to express neural differentiation markers, indicating that the effect of TH in proliferation is linked with differentiation in the nervous system. To date, the molecular mechanism by which TH affects cell proliferation during embryogenesis is unknown. We have utilized the yeast two-hybrid system to identify protein partners of TH that could lead us to define the mechanism or pathway through which TH functions. Using this assay we have identified a new variant of TH designated TH-B, as a potential protein partner of the original TH, now referred to as TH-A. The sequence for TH-B was found to be 85% identical at the amino acid level to the TH-A sequence. Further characterization of the TH-B variant using RT-PCR indicates that it is expressed ubiquitously throughout development from early cleavage stages until at least the tadpole stage. TH-B association with TH-A was confirmed in co-immnoprecipitation studies in Xenopus, indicating that the two variants may function as an oligomer in vivo. These studies reveal the presence of an isoform of TH that may possess novel functional capabilities.

Potential structural role of non-coding and coding RNAs in the organization of the cytoskeleton at the vegetal cortex of Xenopus oocytes.

The localization of RNA within a cell or embryo is crucial for proper cellular function or development. There is evidence that the cytoskeleton and RNA may function in the anchoring of localized RNAs at the vegetal cortex of Xenopus laevis oocytes. We found that the organization of the cytokeratin filaments but not the actin cytoskeleton depends on the presence of intact VegT mRNA and a noncoding RNA, Xlsirts. Destruction of either of these transcripts results in disruption of the cytokeratin cytoskeleton in a transcript-specific manner and interferes with proper formation of the germinal granules and subsequent development of the germline. Analysis of the distribution of endogenous VegT and Xlsirts in live oocytes using molecular beacons showed that these RNAs are integrated into the cytokeratin cytoskeleton. These results demonstrate a novel structural role of coding and noncoding RNAs in the organization of the vegetal cortex of Xenopus oocytes.

Inhibition of the anaphase-promoting complex by the Xnf7 ubiquitin ligase.

Degradation of specific protein substrates by the anaphase-promoting complex/cyclosome (APC) is critical for mitotic exit. We have identified the protein Xenopus nuclear factor 7 (Xnf7) as a novel APC inhibitor able to regulate the timing of exit from mitosis. Immunodepletion of Xnf7 from Xenopus laevis egg extracts accelerated the degradation of APC substrates cyclin B1, cyclin B2, and securin upon release from cytostatic factor arrest, whereas excess Xnf7 inhibited APC activity. Interestingly, Xnf7 exhibited intrinsic ubiquitin ligase activity, and this activity was required for APC inhibition. Unlike other reported APC inhibitors, Xnf7 did not associate with Cdc20, but rather bound directly to core subunits of the APC. Furthermore, Xnf7 was required for spindle assembly checkpoint function in egg extracts. These data suggest that Xnf7 is an APC inhibitor able to link spindle status to the APC through direct association with APC core components.

RNA localization mechanisms in oocytes.

In many animals, normal development depends on the asymmetric distribution of maternal determinants, including various coding and noncoding RNAs, within the oocyte. The temporal and spatial distribution of localized RNAs is determined by intricate mechanisms that regulate their movement and anchoring. These mechanisms involve cis-acting sequences within the RNA molecules and a multitude of trans-acting factors, as well as a polarized cytoskeleton, molecular motors and specific transporting organelles. The latest studies show that the fates of localized RNAs within the oocyte cytoplasm are predetermined in the nucleus and that nuclear proteins, some of them deposited on RNAs during splicing, together with the components of the RNA-silencing pathway, dictate the proper movement, targeting, anchoring and translatability of localized RNAs.

The Xenopus laevis morphogenetic factor, tumorhead, causes defects in polarized growth and cytokinesis in the fission yeast, Schizosaccharomyces pombe.

Tumorhead (TH) is a maternally expressed gene product that regulates neural tube morphogenesis in the amphibian, Xenopus laevis. Here we describe the effects of TH expression in the rod-shaped fission yeast, Schizosaccharomyces pombe. Expression of TH in S. pombe resulted in severe morphological defects, including ovoid, bottle-shaped, and enlarged cells. Multi-septated cells were also observed in TH expressing cultures, indicating that TH is inhibitory to a process required for the completion of cytokinesis. TH expression caused significant actin and microtubule cytoskeletal defects, including depolarization of the cortical F-actin cytoskeleton and increased microtubule formation. Immunostaining experiments showed that TH is localized to the cell cortex, cell tips, and septum in S. pombe cells. Localization of TH to the cell cortex was dependent on the S. pombe PAK homolog, Shk1. Moreover, TH expression was inhibitory to the growth of a mutant defective in Shk1 function, suggesting that TH may interact with a component(s) of a PAK-mediated morphogenetic regulatory pathway in S. pombe. Taken together, our findings demonstrate that S. pombe may be a useful model organism for identifying potential TH interacting factors.

Sm proteins, the constituents of the spliceosome, are components of nuage and mitochondrial cement in Xenopus oocytes.

A conserved feature of germ cells in many animal species is the presence of perinuclear electron-dense material called the "nuage" that is believed to be a precursor of germinal (or polar or P) granules. In Xenopus oogenesis the nuage is first observed near the nuclear envelope and subsequently in close contact with mitochondria, at which stage it is called the mitochondrial cement. In this study, we found that, in Xenopus pre-stage I and stage I oocytes, nuage and mitochondrial cement contain the spliceosomal Sm proteins, Xcat2 mRNA, and DEAD-box RNA helicase XVLG1. Other components of Cajal bodies or splicing machinery such as coilin, SMN protein, and snRNAs are absent from the nuage and mitochondrial cement. We suggest that Xenopus Sm proteins have adapted to a role independent of pre-mRNA splicing and that instead of binding to their traditional spliceosomal partner such as snRNA, they bind mRNAs that are the components of germinal granules (i.e., Xcat2 mRNA) and facilitate the transport of these mRNAs from the nucleus to the nuage that is a precursor of germinal granules. In addition, the presence of Vasa-like DEAD-box helicase in Xenopus nuage suggests involvement of nuage in the microRNA and/or RNAi pathway, similar to the role of nuage in Drosophila.

Formation, architecture and polarity of female germline cyst in Xenopus.

Little is known about the formation of germline cyst and the differentiation of oocyte within the cyst in vertebrates. In the majority of invertebrates in the initial stages of gametogenesis, male and female germ cells develop in full synchrony as a syncytia of interconnected cells called germline cysts (clusters, nests). Using electron microscopy, immunostaining and three-dimensional reconstruction, we were able to elucidate the process of cyst formation in the developing ovary of the vertebrate Xenopus laevis. We found that the germline cyst in Xenopus contains 16 cells that are similar in general architecture and molecular composition to the cyst in Drosophila. Nest cells are connected by cytoplasmic bridges that contain ring canal-like structures. The nest cells contain a structure similar to the Drosophila fusome that that is probably involved in anchoring of the centrioles and organization of the primary mitochondrial cloud (PMC) around the centriole. We also find that in contrast to other organisms, in Xenopus, apoptosis is a rare event within the developing ovary. Our studies indicate that the processes responsible for the formation of female germline cysts and the establishment of germ cell polarity are highly conserved between invertebrates and vertebrates. The dissimilarities between Drosophila and Xenopus and the uniqueness of each system probably evolved through modifications of the same fundamental design of the germline cyst.

Evidence for antagonism of BMP-4 signals by MAP kinase during Xenopus axis determination and neural specification.

We have previously shown that mitogen-activated protein (MAP) kinase activity is required for neural specification in Xenopus. In mammalian cells, the BMP-4 effector Smad1 is inhibited by phosphorylation at MAP kinase sites (Kretzschmar et al., 1997). To test the hypothesis that MAP kinase inhibits the BMP-4/Smad1 pathway during early Xenopus development, we have generated a Smad1 mutant lacking the MAP kinase phosphorylation sites (M4A-Smad1) and compared the effects of wild-type (WT)- and M4A-Smad1 on axial pattern and neural specification in Xenopus embryos. Although overexpression of either WT- or M4A-Smad1 produced ventralized embryos, at each mRNA concentration, M4A-Smad1 had a greater ventralizing effect than WT-Smad1. Interestingly, overexpression of either form of Smad1 in ventral blastomeres disrupted posterior pattern and morphogenesis; again, more severe defects were produced by expression of M4A-Smad1 than by equal amounts of WT-Smad1. Ectodermal expression of M4A-Smad1 disrupted expression of the anterior neural gene otx2 in vivo and inhibited neural specification in response to endogenous signals in mesoderm-ectoderm recombinates. In contrast, overexpression of WT-Smad1 at identical levels had little effect on either neural specification or otx2 expression. Comparisons of protein levels following overexpression of either WT- or M4A-Smad1 indicate that WT-Smad1 may be slightly more stable than M4A-Smad1; thus, differences in stability cannot account for the increased effectiveness of M4A-Smad1. Our results demonstrate that mutations disrupting the MAPK phosphorylation sites act collectively as a gain-of-function mutation in Smad1 and that inhibitory phosphorylation of Smad1 may be a significant mechanism for the regulation of BMP-4/Smad1 signals during Xenopus development.

Identification and characterization of the Xlsirt cis-acting RNA localization element.

There are many RNAs that are localized to the vegetal cortex of Xenopus laevis oocytes. One family of localized transcripts, Xlsirts (Xenopus laevis short interspersed repeat transcripts), are defined by the presence of non-coding repeat units 79-81 nucleotides long. Endogenous Xlsirt RNAs are localized through the METRO (message transport organizer) pathway that localizes RNAs during stages I and II of oogenesis. Interestingly, exogenous Xlsirt RNAs that are injected into oocytes can utilize both the METRO pathway as well as the Late pathway, which localizes RNAs during the late stages of oogenesis (stages III-VI). In all cases thus far analyzed, the localization process relies on the presence of cis-acting elements on the transcripts that are responsible for directing localization. To better understand the mechanism responsible for the use of the METRO and Late localization pathways, we sought to identify pathway-specific cis-acting localization elements contained in Xlsirts. The results showed that an intact 137 nucleotide element was necessary and sufficient to localize RNAs through the METRO and Late pathways. Further analysis of this element identified putative METRO and Late pathway localization sub-elements. Computer analysis relates the secondary structure of the 137 nt element to its ability to function as a localization element.

Apoptotic phosphorylation of histone H2B is mediated by mammalian sterile twenty kinase.

DNA in eukaryotic cells is associated with histone proteins; hence, hallmark properties of apoptosis, such as chromatin condensation, may be regulated by posttranslational histone modifications. Here we report that phosphorylation of histone H2B at serine 14 (S14) correlates with cells undergoing programmed cell death in vertebrates. We identify a 34 kDa apoptosis-induced H2B kinase as caspase-cleaved Mst1 (mammalian sterile twenty) kinase. Mst1 can phosphorylate H2B at S14 in vitro and in vivo, and the onset of H2B S14 phosphorylation is dependent upon cleavage of Mst1 by caspase-3. These data reveal a histone modification that is uniquely associated with apoptotic chromatin in species ranging from frogs to humans and provide insights into a previously unrecognized physiological substrate for Mst1 kinase. Our data provide evidence for a potential apoptotic "histone code."

Identification of new Xlsirt family members in the Xenopus laevis oocyte.

Xenopus laevis short interspersed repeat transcripts (Xlsirts) are a family of noncoding RNAs defined by the presence of a specific repeated sequence that acts as a vegetal localization element. Previous studies have demonstrated that Xlsirts function as localization elements to localize RNA and also in anchoring mRNA at the vegetal cortex. However, the identity of the Xlsirts containing family members present at the cortex was unknown. We identified 17 new Xlsirt cDNAs from an oocyte cDNA library. In addition to being associated with noncoding sequences, the repeats were also present in cDNAs with open reading frames. Xlsirt RNAs with repeats in the correct orientation were capable of localizing to the vegetal cortex. Our observations demonstrate that a heterogeneous population of Xlsirt RNAs is present at the cortex and that this population contains both noncoding RNAs and RNAs encoding proteins that are likely to play important roles in the subsequent development of the embryo.

Difference in the maternal and zygotic contributions of tumorhead on embryogenesis.

Tumorhead (TH) is a maternally expressed gene in Xenopus laevis, that when overexpressed, increased proliferation of ectodermal derivatives and inhibited neural and epidermal differentiation. However, injection of anti-TH antibodies inhibited cleavage of all blastomeres, not only those contributing to the ectoderm. The injection of TH morpholino antisense oligonucleotide (TH-MO), which inhibits translation of TH mRNA, did not affect early cleavage but inhibited cell division in both the neural field and epidermis. This was accompanied by the inhibition of neural and epidermal markers. TH-MO did not affect the formation and differentiation of mesoderm and endoderm derivatives. Our overexpression and loss-of-function studies demonstrated that TH plays an important role in differentiation of the ectoderm by regulating cell proliferation. They also supported the conclusion that the maternal component of TH may affect the cell cycle in all cells, while the zygotic component has a germ layer-specific effect on the ectoderm.