Endocytic Machinery Protein SlaB Is Dispensable for Polarity Establishment but Necessary for Polarity Maintenance in Hyphal Tip Cells of Aspergillus nidulans

The Aspergillus nidulans endocytic internalization protein SlaB is essential, in agreement with the key role in apical extension attributed to endocytosis. We constructed, by gene replacement, a nitrate-inducible, ammonium-repressible slaB1 allele for conditional SlaB expression. Video microscopy showed that repressed slaB1 cells are able to establish but unable to maintain a stable polarity axis, arresting growth with budding-yeast-like morphology shortly after initially normal germ tube emergence. Using green fluorescent protein (GFP)-tagged secretory v-SNARE SynA, which continuously recycles to the plasma membrane after being efficiently endocytosed, we establish that SlaB is crucial for endocytosis, although it is dispensable for the anterograde traffic of SynA and of the t-SNARE Pep12 to the plasma and vacuolar membrane, respectively. By confocal microscopy, repressed slaB1 germlings show deep plasma membrane invaginations. Ammonium-to-nitrate medium shift experiments demonstrated reversibility of the null polarity maintenance phenotype and correlation of normal apical extension with resumption of SynA endocytosis. In contrast, SlaB downregulation in hyphae that had progressed far beyond germ tube emergence led to marked polarity maintenance defects correlating with deficient SynA endocytosis. Thus, the strict correlation between abolishment of endocytosis and disability of polarity maintenance that we report here supports the view that hyphal growth requires coupling of secretion and endocytosis. However, downregulated slaB1 cells form F-actin clumps containing the actin-binding protein AbpA, and thus F-actin misregulation cannot be completely disregarded as a possible contributor to defective apical extension. Latrunculin B treatment of SlaB-downregulated tips reduced the formation of AbpA clumps without promoting growth and revealed the formation of cortical “comets” of AbpA.Germinating asexual spores (conidiospores) of Aspergillus nidulans transiently undergo isotropic growth (“swelling”) before establishing a polarity axis that grows by apical extension, leading to the characteristic tubular morphology of the fungal cell (15, 16, 33). Stable maintenance of a polarity axis at the high apical extension rates of A. nidulans (∼0.5 μm/min at 25°C) (23) can be attributable, at least in part, to the polarization of the secretory apparatus and the predominant and highly efficient delivery of secretory vesicles to the apex (8, 18, 40, 49). In addition, work from several laboratories strongly indicated that hyphal tip growth also involves endocytosis. A key observation supporting this involvement was that despite the fact that endocytosis can occur elsewhere, the endocytic internalization machinery predominates in the hyphal tip, forming a subapical collar. The spatial association of this collar with the apical region where secretory materials are delivered would allow removal of excess lipids/proteins reaching the plasma membrane with secretory vesicles (1, 2, 30, 49, 51, 57), but, most importantly, rapid endocytic recycling (i.e., efficient endocytosis of membrane proteins followed by their redelivery to the plasma membrane) can generate and maintain polarity, as shown with the v-SNARE and secretory-vesicle-resident SynA, which is a substrate of the subapical endocytic ring (1, 49, 52). It is plausible that such a mechanism could drive the polarization of one or more proteins acting as positional cues to mediate polarity maintenance.Genetic evidence strongly supported the conclusion that endocytosis is required for apical extension. Mutational inactivation of the A. nidulans fimbrin FimA or of the small GTPase ArfB^Arf6 (a regulator of endocytosis from fungi to mammals), led to delayed polarity establishment and morphologically aberrant tips indicative of polarity maintenance defects (30, 51). These mutations, although very severely debilitating, are not lethal. In contrast, heterokaryon rescue showed that SlaB, a key F-actin regulator of the endocytic internalization machinery (26), is essential in A. nidulans (2). slaBΔ cells are able to establish polarity, but they arrest in apical extension during the very early steps of polarity maintenance with a bud-like germ tube (2). However, work with Aspergillus oryzae using a thiamine-repressible promoter to drive A. oryzae End4 (AoEnd4) (SlaB) expression showed that although endocytosis was prevented and hyphal morphology altered under repressing conditions, hyphal tip extension and polarity maintenance were not completely abolished (20), perhaps suggesting that the phenotype of the thiamine-repressed conditional allele might not fully resemble the null phenotype.F-actin strongly predominates in the hyphal tips (2, 14, 17, 49, 51). Because endocytic internalization is powered by F-actin (27), predominance of endocytic “patches” (i.e., sites of endocytic internalization) in the tip accounts, at least in part, for F-actin polarization. However, F-actin plays fundamental nonendocytic roles in the tip; for example, actin cables nucleated by the SepA formin localizing to the apical crescent are thought to play a major role in secretion, whereas a network of F-actin microfilaments appears to be a major component of the Spitzenkörper (4, 21, 43, 49). Notably, all genes used to address the role of endocytosis in apical extension share in common their involvement in regulating F-actin. Thus, the Saccharomyces cerevisiae ArfB orthologue Arf3p regulates endocytosis but also appears to regulate F-actin at multiple levels (12, 28, 44). Like their respective S. cerevisiae orthologues Sla2p and Sac6p, SlaB and FimA are key components of endocytic patches, but in budding yeast their orthologues appear to regulate F-actin dynamics beyond endocytosis (27, 35, 56).To gain insight into the essential role of SlaB in A. nidulans, we designed a conditional expression allele. Time-lapse microscopy under restrictive conditions demonstrated that polarity establishment is essentially normal but that these mutant germ tubes arrested in apical extension subsequently undergo swelling, acquiring the characteristic bud-like shape of abortive slaBΔ germlings. Medium shift experiments allowed us to address the role of SlaB in apical extension beyond these early stages of polarity maintenance. We demonstrate the key role that SlaB plays in endocytosis and polarity maintenance, but we also show that deficiency of SlaB affects the actin cytoskeleton.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Linkage of Oxidative Stress and Mitochondrial Dysfunctions to Spontaneous Culture Degeneration in Aspergillus nidulans

Filamentous fungi including mushrooms frequently and spontaneously degenerate during subsequent culture maintenance on artificial media, which shows the loss or reduction abilities of asexual sporulation, sexuality, fruiting, and production of secondary metabolites, thus leading to economic losses during mass production. To better understand the underlying mechanisms of fungal degeneration, the model fungus Aspergillus nidulans was employed in this study for comprehensive analyses. First, linkage of oxidative stress to culture degeneration was evident in A. nidulans. Taken together with the verifications of cell biology and biochemical data, a comparative mitochondrial proteome analysis revealed that, unlike the healthy wild type, a spontaneous fluffy sector culture of A. nidulans demonstrated the characteristics of mitochondrial dysfunctions. Relative to the wild type, the features of cytochrome c release, calcium overload and up-regulation of apoptosis inducing factors evident in sector mitochondria suggested a linkage of fungal degeneration to cell apoptosis. However, the sector culture could still be maintained for generations without the signs of growth arrest. Up-regulation of the heat shock protein chaperones, anti-apoptotic factors and DNA repair proteins in the sector could account for the compromise in cell death. The results of this study not only shed new lights on the mechanisms of spontaneous degeneration of fungal cultures but will also provide alternative biomarkers to monitor fungal culture degeneration.Culture degeneration, also called colony deterioration, of filamentous fungi can frequently occur during subsequent maintenance of fungal culture on artificial media by showing morphological changes, such as colony sectorization, loss or impaired ability of sporulation, fruiting, and sexuality (1, 2). It was first called as “woolly degeneration” in the model fungus Neurospora crassa (3). These morphological variations are also accompanied with the loss or reduction in secondary metabolites production, thus resulting in great commercial losses (4–7). Different from the mutation of genes involved in conidiation producing fluffy phenotypes (8–10), fungal culture degeneration spontaneously occurs and is usually irreversible. The rate of colony deterioration varies considerably among fungal species/strains and correlates with the growth environments, especially the composition of nutrient medium (11, 12). In addition, unlike the phenotype of senescence observed in the model fungus Podospora anserina (13), morphological variants of other filamentous fungi could be subcultured for many generations without growth arrest (14, 15). The mechanism(s) underlying fungal culture degeneration is poorly understood. Genetic mutations were not evident in the degenerated fungal isolates of Penicillium chrysogenum (6). Otherwise, methylation of genomic DNA was reported in a sector of the plant pathogenic fungus Fusarium oxysporum after successive subculturing (16). In some fungi, degeneration was linked to chromosome instability (4, 17).Our previous studies on the insect pathogenic fungus Metarhizium anisopliae revealed that fungal culture degeneration showed the signs of aging such as cellular accumulation of reactive oxygen species (ROS)¹, mitochondrial (mt) dysfunctions, and mtDNA glycation (14, 15). According to the vicious cycle theory of aging, mitochondria are the primary source of intracellular ROS production and also one of the important targets of ROS damage, which leads to generation of additional ROS (18, 19). Mitochondrial proteomics analyses have been frequently performed for studies on human diseases, and the physiologies of plants and yeasts (20, 21). Proteins localized in the mitochondria control mt dynamics, morphology, and function and their dysregulation or damage may induce abnormality in mitochondrial function (22). However, it is not known whether these changes occur in degenerated fungal cultures.In this study, cell biology, biochemical and comparative mitochondrial proteomic analyses were performed by using the model fungus Aspergillus nidulans to better understand the features and mechanisms of fungal culture degeneration. We found a significant difference in mitochondrial protein profiles between the wild type (WT) and nonsporulation sector culture. Many of the altered proteins fall into the functional categories of energy metabolism, stress responses and cell death. Functional consequences of these changes are supported by our experimental data. The observed features such as cellular oxidative stress, mitochondrial dysfunctions, accelerated autophagy and releases of apoptotic factors in degenerated A. nidulans resemble the apoptotic process observed in mammalian cells.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Vinyl Sulfones as Antiparasitic Agents and a Structural Basis for Drug Design

Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs.Sleeping sickness (African trypanosomiasis), caused by Trypanosoma brucei, and malaria, caused by Plasmodium falciparum, are significant, parasitic diseases of sub-Saharan Africa (1). Chagas'' disease (South American trypanosomiasis), caused by Trypanosoma cruzi, affects approximately, 16–18 million people in South and Central America. For all three of these protozoan diseases, resistance and toxicity to current therapies makes treatment increasingly problematic, and thus the development of new drugs is an important priority (2–4).T. cruzi, T. brucei, and P. falciparum produce an array of potential target enzymes implicated in pathogenesis and host cell invasion, including a number of essential and closely related papain-family cysteine proteases (5, 6). Inhibitors of cruzain and rhodesain, major cathepsin L-like papain-family cysteine proteases of T. cruzi and T. brucei rhodesiense (7–10) display considerable antitrypanosomal activity (11, 12), and some classes have been shown to cure T. cruzi infection in mouse models (11, 13, 14).In P. falciparum, the papain-family cysteine proteases falcipain-2 (FP-2)⁶ and falcipain-3 (FP-3) are known to catalyze the proteolysis of host hemoglobin, a process that is essential for the development of erythrocytic parasites (15–17). Specific inhibitors, targeted to both enzymes, display antiplasmodial activity (18). However, although the abnormal phenotype of FP-2 knock-outs is “rescued” during later stages of trophozoite development (17), FP-3 has proved recalcitrant to gene knock-out (16) suggesting a critical function for this enzyme and underscoring its potential as a drug target.Sequence analyses and substrate profiling identify cruzain, rhodesain, and FP-3 as cathepsin L-like, and several studies describe classes of small molecule inhibitors that target multiple cathepsin L-like cysteine proteases, some with overlapping antiparasitic activity (19–22). Among these small molecules, vinyl sulfones have been shown to be effective inhibitors of a number of papain family-like cysteine proteases (19, 23–27). Vinyl sulfones have many desirable attributes, including selectivity for cysteine proteases over serine proteases, stable inactivation of the target enzyme, and relative inertness in the absence of the protease target active site (25). This class has also been shown to have desirable pharmacokinetic and safety profiles in rodents, dogs, and primates (28, 29). We have determined the crystal structures of cruzain, rhodesain, and FP-3 bound to vinyl sulfone inhibitors and performed inhibition kinetics for each enzyme. Our results highlight key areas of interaction between proteases and inhibitors. These results help validate the vinyl sulfones as a class of antiparasitic drugs and provide structural insights to facilitate the design or modification of other small molecule inhibitor scaffolds.