Knockdown of CENH3 in Arabidopsis reduces mitotic divisions and causes sterility by disturbed meiotic chromosome segregation

The histone H3 variant (CENH3) of centromeric nucleosomes is essential for kinetochore assembly and thus for chromosome segregation in eukaryotes. The mechanism(s) that determine centromere identity, assembly and maintenance of kinetochores are still poorly understood. Although the role of CENH3 during mitosis has been studied in several organisms, little is known about its meiotic function. We show that RNAi-mediated CENH3 knockdown in Arabidopsis thaliana caused dwarfism as the result of a reduced number of mitotic divisions. The remaining mitotic divisions appeared to be error-free. CENH3 RNAi transformants had reduced fertility because of frequently disturbed meiotic chromosome segregation. N-terminally truncated EYFP-CENH3(C) is deposited to and functional within Arabidopsis centromeres of mitotic chromosomes, but cannot be loaded onto centromeres of meiotic nuclei. Thus the N-terminal part is apparently required for CENH3 loading during meiosis. EYFP-CENH3(C) expression reduces the amount of endogenous CENH3, thus mimicking the effect of RNAi. The consequences of reduced endogenous CENH3 and lack of meiotic incorporation of EYFP-CENH3(C) are reduced fertility caused by insufficient CENH3 loading to the centromeres of meiotic chromosomes, subsequent lagging of chromosomes and formation of micronuclei.     

Three routes to suppression of the neurodegenerative phenotypes caused by Kinesin heavy chain mutations

Kinesin-1 is a motor protein that moves stepwise along microtubules by employing dimerized kinesin heavy chain (Khc) subunits that alternate cycles of microtubule binding, conformational change, and ATP hydrolysis. Mutations in the Drosophila Khc gene are known to cause distal paralysis and lethality preceded by the occurrence of dystrophic axon terminals, reduced axonal transport, organelle-filled axonal swellings, and impaired action potential propagation. Mutations in the equivalent human gene, Kif5A, result in similar problems that cause hereditary spastic paraplegia (HSP) and Charcot-Marie-Tooth type 2 (CMT2) distal neuropathies. By comparing the phenotypes and the complementation behaviors of a large set of Khc missense alleles, including one that is identical to a human Kif5A HSP allele, we identified three routes to suppression of Khc phenotypes: nutrient restriction, genetic background manipulation, and a remarkable intramolecular complementation between mutations known or likely to cause reciprocal changes in the rate of microtubule-stimulated ADP release by kinesin-1. Our results reveal the value of large-scale complementation analysis for gaining insight into protein structure-function relationships in vivo and point to possible paths for suppressing symptoms of HSP and related distal neuropathies.     

A New C-Terminal Cleavage Product of Procaspase-8, p30, Defines an Alternative Pathway of Procaspase-8 Activation

Caspase-8 is the main initiator caspase in death receptor-induced apoptosis. Procaspase-8 is activated at the death-inducing signaling complex (DISC). Previous studies suggested a two-step model of procaspase-8 activation. The first cleavage step occurs between the protease domains p18 and p10. The second cleavage step takes place between the prodomain and the large protease subunit (p18). Subsequently, the active caspase-8 heterotetramer p18₂-p10₂ is released into the cytosol, starting the apoptotic signaling cascade. In this report, we have further analyzed procaspase-8 processing upon death receptor stimulation directly at the DISC and in the cytosol. We have found an alternative sequence of cleavage events for procaspase-8. We have demonstrated that the first cleavage can also occur between the prodomain and the large protease subunit (p18). The resulting cleavage product, p30, contains both the large protease subunit (p18) and the small protease subunit (p10). p30 is further processed to p10 and p18 by active caspases. Furthermore, we show that p30 can sensitize cells toward death receptor-induced apoptosis. Taken together, our data suggest an alternative mechanism of procaspase-8 activation at the DISC.Apoptosis can be triggered by a number of factors, including UV or γ-irradiation, chemotherapeutic drugs, and signaling from death receptors (11, 12). CD95 (APO-1/Fas) is a member of the death receptor family, a subfamily of the tumor necrosis factor receptor (TNF-R) superfamily (1, 30). Eight members of the death receptor subfamily have been characterized so far: TNF-R1 (DR1, CD120a, p55, p60), CD95 (DR2, APO-1, Fas), DR3 (APO-3, LARD, TRAMP, WSL1), TRAIL-R1 (APO-2, DR4), TRAIL-R2 (DR5, KILLER, TRICK2), DR6, EDA-R, and NGF-R (13). Cross-linking of CD95 by its natural ligand, CD95L (CD178) (29), or by agonistic antibodies induces apoptosis in sensitive cells (31, 36). The death-inducing signaling complex (DISC) is formed within seconds after CD95 stimulation (9). The DISC consists of oligomerized, probably trimerized CD95 receptors, the adaptor molecule FADD, two isoforms of procaspase-8 (procaspase-8a and -8b), procaspase-10, and c-FLIP_L/S/R (6, 19, 21, 25, 27). The interactions between molecules at the DISC are based on homotypic contacts. The death domain of the receptor interacts with the death domain of FADD, while the death effector domain (DED) of FADD interacts with the N-terminal tandem DEDs of procaspase-8 and -10 and c-FLIP_L/S/R.Two isoforms of procaspase-8 (procaspase-8a and procaspase-8b) were reported to be bound to the DISC (24). Both isoforms possess two tandem DEDs, as well as the catalytic subunits p18 and p10 (see Fig. ​Fig.1A).1A). Procaspase-8a contains an additional 2-kDa (15-amino-acid [aa]) fragment, which results from the translation of exon 9. This small fragment is located between the second DED and the large catalytic subunit, resulting in different lengths of procaspase-8a and -8b (p55 and p53 kDa), respectively.Open in a separate windowFIG. 1.A new 30-kDa protein is detected by the anti-caspase-8 MAb C15. (A) Scheme of procaspase-8 and its cleavage products. The binding sites of the anti-caspase-8 MAbs C5 and C15 are indicated. (B) The B-lymphoblastoid cell lines SKW6.4, Raji, and BJAB and the T-cell lines CEM, Jurkat 16, and caspase-8-deficient Jurkat (clone JI9.2) were stimulated with LZ-CD95L for the indicated times, followed by caspase-8 immunoprecipitation (C8-IP) using the anti-caspase-8 MAb C15 directed against the p18 subunit of procaspase-8. Western blotting of immunoprecipitates was performed using the anti-caspase-8 MAb C15 (**, Ig heavy chain; *, unspecific band). (C) SKW6.4 cells were stimulated with LZ-CD95L for different times, and procaspase-8 processing in total cellular lysates was analyzed by Western blotting using the anti-caspase-8 MAb C15. (D) B-lymphoblastoid BJAB cells were stimulated with LZ-TRAIL for different times, and procaspase-8 processing was analyzed as described for panel C. (E) Primary human T cells (day 6) were stimulated with LZ-CD95L, and procaspase-8 processing was analyzed as described for panel C (*, unspecific band).Activation of procaspase-8 is believed to follow an “induced-proximity” model in which high local concentrations and a favorable mutual orientation of procaspase-8 molecules at the DISC lead to their autoproteolytic processing (2, 3, 20). There is strong evidence from several in vitro studies that autoproteolytic activation of procaspase-8 occurs after oligomerization at the receptor complex (20). Furthermore, it has been shown that homodimers of procaspase-8 have proteolytic activity and that proteolytic processing of procaspase-8 occurs between precursor homodimers (3).Procaspase-8a/b (p55/p53) processing at the DISC has been described to involve two sequential cleavage steps (see Fig. ​Fig.1A).1A). This process is referred to as the “two-step model” (3, 17). The first cleavage step occurs between the two protease domains, and the second cleavage step takes place between the prodomain and the large protease subunit (see Fig. ​Fig.1A)1A) (15). During the first cleavage step, the cleavage at Asp³⁷⁴ generates the two subunits p43/p41 and p12. Both cleavage products remain bound to the DISC: p43/p41 by DED interactions and p12 by interactions with the large protease domain of p43/p41. The second cleavage step takes place at Asp²¹⁶ and Asp³⁸⁴, producing the active enzyme subunits p18, p10, and the prodomain p26/p24. As a result of procaspase-8 processing, the active caspase-8 heterotetramer p18₂-p10₂ is formed at the DISC. This heterotetramer is subsequently released into the cytosol, starting the apoptotic signaling cascade (14).Recent studies have shown that processing of procaspase-8 at the DISC is more complicated and can involve additional steps like the generation of a prolonged prodomain of procaspase-8, termed CAP3 (p27), that is quickly converted to p26 (see Fig. ​Fig.1A)1A) (7).In addition to its central role in death receptor-induced apoptosis, caspase-8 was reported to be required for proliferation of lymphocytes (12, 23). Recently caspase-8 was shown to be an important factor for NF-κB activation following T-cell receptor stimulation (28). The mechanism underlying the dual role of caspase-8 activity and its regulation is largely unknown.In the present study, we show that upon death receptor stimulation, p30 is formed by cleavage at Asp²¹⁰, a yet-unknown cleavage product of procaspase-8, which comprises the C terminus of procaspase-8. p30 turned out to be a key intermediate product in the course of procaspase-8 processing. Furthermore, we suggest that the p30-mediated activation of procaspase-8 plays an important role in the amplification of the death signal. Taken together, our findings provide a new mechanism of procaspase-8 activation and extend the current two-step cleavage model by an alternative activation pathway.     

Multi-species sequence comparison: the next frontier in genome annotation

Multi-species comparisons of DNA sequences are more powerful for discovering functional sequences than pairwise DNA sequence comparisons. Most current computational tools have been designed for pairwise comparisons, and efficient extension of these tools to multiple species will require knowledge of the ideal evolutionary distance to choose and the development of new algorithms for alignment, analysis of conservation, and visualization of results.     

Pre-clinical and clinical significance of heparanase in Ewing's sarcoma

Heparanase is an endoglycosidase that specifically cleaves heparan sulphate side chains of heparan sulphate proteoglycans, activity that is strongly implicated in cell migration and invasion associated with tumour metastasis, angiogenesis and inflammation. Heparanase up-regulation was documented in an increasing number of human carcinomas, correlating with reduced post-operative survival rate and enhanced tumour angiogenesis. Expression and significance of heparanase in human sarcomas has not been so far reported. Here, we applied the Ewing's sarcoma cell line TC71 and demonstrated a potent inhibition of cell invasion in vitro and tumour xenograft growth in vivo upon treatment with a specific inhibitor of heparanase enzymatic activity (compound SST0001, non-anticoagulant N-acetylated, glycol split heparin). Next, we examined heparanase expression and cellular localization by immunostaining of a cohort of 69 patients diagnosed with Ewing's sarcoma. Heparanase staining was noted in all patients. Notably, heparanase staining intensity correlated with increased tumour size (P = 0.04) and with patients' age (P = 0.03), two prognostic factors associated with a worse outcome. Our study indicates that heparanase expression is induced in Ewing's sarcoma and associates with poor prognosis. Moreover, it encourages the inclusion of heparanase inhibitors (i.e. SST0001) in newly developed therapeutic modalities directed against Ewing's sarcoma and likely other malignancies.     

Enhancement of synaptic plasticity through chronically reduced Ca2+ flux during uncorrelated activity

The plasticity of synapses within neural circuits is regulated by activity, but the underlying mechanisms remain elusive. Using the dye FM1-43 to directly image presynaptic function, we found that large numbers of presynaptic terminals in hippocampal cultures have a low release probability. While these terminals were not readily modifiable, a transient but not permanent long-term reduction of network activity or Ca2+ influx could increase their modifiability. This modulation of plasticity was mediated by Ca2+ flux through NMDA and voltage-gated calcium channels and was lost within 48 hr. A more permanent enhancement of synaptic plasticity was achieved by selectively reducing the Ca2+ flux associated with uncorrelated activity via adjustment of the voltage-dependent Mg2+ block of the NMDAR. Upregulation of NR2B-containing NMDARs induced by this treatment is an important but not sole contributor to the enhancement of plasticity. Thus, quantity and quality of activity have differential effects on the intrinsic plasticity of neurons.     

Phylo-VISTA: interactive visualization of multiple DNA sequence alignments

MOTIVATION: The power of multi-sequence comparison for biological discovery is well established. The need for new capabilities to visualize and compare cross-species alignment data is intensified by the growing number of genomic sequence datasets being generated for an ever-increasing number of organisms. To be efficient these visualization algorithms must support the ability to accommodate consistently a wide range of evolutionary distances in a comparison framework based upon phylogenetic relationships. RESULTS: We have developed Phylo-VISTA, an interactive tool for analyzing multiple alignments by visualizing a similarity measure for multiple DNA sequences. The complexity of visual presentation is effectively organized using a framework based upon interspecies phylogenetic relationships. The phylogenetic organization supports rapid, user-guided interspecies comparison. To aid in navigation through large sequence datasets, Phylo-VISTA leverages concepts from VISTA that provide a user with the ability to select and view data at varying resolutions. The combination of multiresolution data visualization and analysis, combined with the phylogenetic framework for interspecies comparison, produces a highly flexible and powerful tool for visual data analysis of multiple sequence alignments. AVAILABILITY: Phylo-VISTA is available at http://www-gsd.lbl.gov/phylovista. It requires an Internet browser with Java Plug-in 1.4.2 and it is integrated into the global alignment program LAGAN at http://lagan.stanford.edu