F-Actin Dynamics in Neurospora crassa

This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.Actins are highly conserved proteins found in all eukaryotes and have an enormous variety of cellular roles. The monomeric form (globular actin, or G-actin) can self-assemble, with the aid of numerous actin-binding proteins (ABPs), into microfilaments (filamentous actin, or F-actin), which, together with microtubules, form the two major components of the fungal cytoskeleton. Numerous pharmacological and genetic studies of fungi have demonstrated crucial roles for F-actin in cell polarity, exocytosis, endocytosis, cytokinesis, and organelle movement (6, 7, 20, 34, 35, 51, 52, 59). Phalloidin staining, immunofluorescent labeling, and fluorescent-protein (FP)-based live-cell imaging have revealed three distinct subpopulations of F-actin-containing structures in fungi: patches, cables, and rings (1, 14, 28, 34, 60, 63, 64). Actin patches are associated with the plasma membrane and represent an accumulation of F-actin around endocytic vesicles (3, 26, 57). Actin cables are bundles of actin filaments stabilized with cross-linking proteins, such as tropomyosins and fimbrin, and are assembled by formins at sites of active growth, where they form tracks for myosin V-dependent polarized secretion and organelle transport (10, 16, 17, 27, 38, 47, 48). Cables, unlike patches, are absolutely required for polarized growth in the budding yeast Saccharomyces cerevisiae (34, 38). Contractile actomyosin rings are essential for cytokinesis in budding yeast, whereas in filamentous fungi, actin rings are less well studied but are known to be involved in septum formation (20, 28, 34, 39, 40).Actin cables and patches have been particularly well studied in budding yeast. However, there are likely to be important differences between F-actin architecture and dynamics in budding yeast and those in filamentous fungi, as budding yeasts display only a short period of polarized growth during bud formation, which is followed by isotropic growth over the bud surface (10). Sustained polarized growth during hyphal morphogenesis is a defining feature of filamentous fungi (21), making them attractive models for studying the roles of the actin cytoskeleton in cell polarization, tip growth, and organelle transport.In Neurospora crassa and other filamentous fungi, disruption of the actin cytoskeleton leads to rapid tip swelling, which indicates perturbation of polarized tip growth, demonstrating a critical role for F-actin in targeted secretion to particular sites on the plasma membrane (7, 22, 29, 56). Immunofluorescence studies of N. crassa have shown that F-actin localizes to hyphal tips as “clouds” and “plaques” (7, 54, 59). However, immunolabeling has failed to reveal actin cables in N. crassa and offers limited insights into F-actin dynamics. Live-cell imaging of F-actin architecture and dynamics has not been accomplished in N. crassa, yet it is expected to yield key insights into cell polarization, tip growth, and intracellular transport.We took advantage of a recently developed live-cell imaging probe for F-actin called Lifeact (43). Lifeact is a 17-amino-acid peptide derived from the N terminus of the budding yeast actin-binding protein Abp140 (5, 63) and has recently been demonstrated to be a universal live-cell imaging marker for F-actin in eukaryotes (43). Here, we report the successful application of fluorescent Lifeact fusion constructs for live-cell imaging of F-actin in N. crassa. We constructed two synthetic genes consisting of Lifeact fused to “synthetic” green fluorescent protein (sGFP) (S65T) (henceforth termed GFP) (12) or red fluorescent protein (TagRFP) (33) and expressed these constructs in various N. crassa strains. In all strain backgrounds, fluorescent Lifeact constructs clearly labeled actin patches, cables, and rings and revealed a direct association of F-actin structures with sites of cell polarization and active tip growth. Our results demonstrate the efficacy of Lifeact as a nontoxic live-cell imaging probe in N. crassa.     

Structure and Function of Glycosylated Tandem Repeats from Candida albicans Als Adhesins

Tandem repeat (TR) regions are common in yeast adhesins, but their structures are unknown, and their activities are poorly understood. TR regions in Candida albicans Als proteins are conserved glycosylated 36-residue sequences with cell-cell aggregation activity (J. M. Rauceo, R. De Armond, H. Otoo, P. C. Kahn, S. A. Klotz, N. K. Gaur, and P. N. Lipke, Eukaryot. Cell 5:1664–1673, 2006). Ab initio modeling with either Rosetta or LINUS generated consistent structures of three-stranded antiparallel β-sheet domains, whereas randomly shuffled sequences with the same composition generated various structures with consistently higher energies. O- and N-glycosylation patterns showed that each TR domain had exposed hydrophobic surfaces surrounded by glycosylation sites. These structures are consistent with domain dimensions and stability measurements by atomic force microscopy (D. Alsteen, V. Dupres, S. A. Klotz, N. K. Gaur, P. N. Lipke, and Y. F. Dufrene, ACS Nano 3:1677–1682, 2009) and with circular dichroism determination of secondary structure and thermal stability. Functional assays showed that the hydrophobic surfaces of TR domains supported binding to polystyrene surfaces and other TR domains, leading to nonsaturable homophilic binding. The domain structures are like “classic” subunit interaction surfaces and can explain previously observed patterns of promiscuous interactions between TR domains in any Als proteins or between TR domains and surfaces of other proteins. Together, the modeling techniques and the supporting data lead to an approach that relates structure and function in many kinds of repeat domains in fungal adhesins.Yeast adhesins are a diverse set of cell adhesion proteins that mediate adhesion to host cells, environmental substrates, other fungi, and coinfecting bacteria (6, 8, 20, 21, 23, 29). The adhesins share common features, including compact N-terminal domains similar to Ig or lectin domains, Thr-rich midpieces, often in tandem repeats, and long highly glycosylated Ser/Thr-rich C-terminal regions that extend the functional domains out from the cell surface. No structures for the Thr-rich midpieces are known, but they can mediate aggregation of fungal cells (33, 35, 47). The prevalence and conservation of such repeats argue that they are functionally important, despite limited data on their structure and function.In Candida albicans, the Als adhesins are homologous proteins, products of 8 loci that encode numerous alleles of cell surface adhesins (16). In each mature Als protein, there are, from the N terminus, three tandem Ig-like domains, a β-sheet-rich conserved 127-residue amyloid-forming T region, a variable number of 36-residue tandem repeats (TRs), and a highly glycosylated stalk region that extends the N-terminal domains away from the cell surface (Fig. 1) (16, 33, 41). The C termini of these and other wall-associated adhesins are covalently cross-linked into the cell wall through transglycosylation of a modified glycosylphosphatidylinositol (GPI) anchor (18, 25). This modular design, including tandem repeats, is typical of fungal adhesins (8).Open in a separate windowFig. 1.Schematic diagram of the sequence of Als5p. The regions are named above, and the number of amino acid residues in each region is shown below. The modeled sequences are in the TR region.The Als protein Ig-like region, T region, and TR region all have protein-protein interaction activities (26, 33, 35). The Ig-like regions can interact with diverse mammalian proteins, presumably in a way analogous to antibody-antigen binding, as has been shown in the homologous protein α-agglutinin from Saccharomyces cerevisiae (8, 24, 26, 35). The T regions interact through formation of amyloid-like structures both in vivo and in vitro (33, 34a, 36). An insight into the function of the tandem repeats followed from observations that Als proteins initiate and maintain cell-to-cell aggregations, either spontaneously (“autoaggregation”) or following adhesion to a bead-bound defined ligand (10, 11, 36). Aggregation is more extensive for Als proteins with more tandem repeats (26, 35). This result suggested that the tandem repeats are uniquely structured to facilitate or mediate the aggregative function. Circular dichroism spectroscopy of the TR region of Als5p shows a β-sheet-rich structure in the soluble protein (35).In support of their direct involvement in aggregation, the repeat region of the C. albicans adhesin Als5p mediates cell-cell aggregation in the absence of the Ig-like and T domains (35). Moreover, the repeats can also potentiate binding of Als5p to fibronectin (35). Thus, the TR domains mediate cellular aggregation and increased binding to fibronectin. In addition, TR domains and their amino acid sequences are highly conserved across several Candida species (3). These properties need to be explained by their three-dimensional structure.Because there are no homologous structures known, we modeled by two independent ab initio methods. Rosetta assembles structures by combining short peptide structures extracted from the protein structural database PDB (38), then combines structures in a Monte Carlo approach, and assesses energetics of assembled structures. Rosetta has recently been shown to generate accurate models for protein-sized domains (40). We also predicted structures with LINUS, which generates randomized structures and rapidly estimates energetics to choose low-energy models (45). The models were supported by structural analyses with atomic force microscopy and circular dichroism spectroscopy. Functional assays showed that the TR domains can mediate binding activities predicted from the calculated structures.     

Diversity Within the O-linked Protein Glycosylation Systems of Acinetobacter Species

The opportunistic human pathogen Acinetobacter baumannii is a concern to health care systems worldwide because of its persistence in clinical settings and the growing frequency of multiple drug resistant infections. To combat this threat, it is necessary to understand factors associated with disease and environmental persistence of A. baumannii. Recently, it was shown that a single biosynthetic pathway was responsible for the generation of capsule polysaccharide and O-linked protein glycosylation. Because of the requirement of these carbohydrates for virulence and the non-template driven nature of glycan biogenesis we investigated the composition, diversity, and properties of the Acinetobacter glycoproteome. Utilizing global and targeted mass spectrometry methods, we examined 15 strains and found extensive glycan diversity in the O-linked glycoproteome of Acinetobacter. Comparison of the 26 glycoproteins identified revealed that different A. baumannii strains target similar protein substrates, both in characteristics of the sites of O-glycosylation and protein identity. Surprisingly, glycan micro-heterogeneity was also observed within nearly all isolates examined demonstrating glycan heterogeneity is a widespread phenomena in Acinetobacter O-linked glycosylation. By comparing the 11 main glycoforms and over 20 alternative glycoforms characterized within the 15 strains, trends within the glycan utilized for O-linked glycosylation could be observed. These trends reveal Acinetobacter O-linked glycosylation favors short (three to five residue) glycans with limited branching containing negatively charged sugars such as GlcNAc3NAcA4OAc or legionaminic/pseudaminic acid derivatives. These observations suggest that although highly diverse, the capsule/O-linked glycan biosynthetic pathways generate glycans with similar characteristics across all A. baumannii.Acinetobacter baumannii is an emerging opportunistic pathogen of increasing significance to health care institutions worldwide (1–3). The growing number of identified multiple drug resistant (MDR)¹ strains (2–4), the ability of isolates to rapidly acquire resistance (3, 4), and the propensity of this agent to survive harsh environmental conditions (5) account for the increasing number of outbreaks in intensive care, burn, or high dependence health care units since the 1970s (2–5). The burden on the global health care system of MDR A. baumannii is further exacerbated by standard infection control measures often being insufficient to quell the spread of A. baumannii to high risk individuals and generally failing to remove A. baumannii from health care institutions (5). Because of these concerns, there is an urgent need to identify strategies to control A. baumannii as well as understand the mechanisms that enable its persistence in health care environments.Surface glycans have been identified as key virulence factors related to persistence and virulence within the clinical setting (6–8). Acinetobacter surface carbohydrates were first identified and studied in A. venetianus strain RAG-1, leading to the identification of a gene locus required for synthesis and export of the surface carbohydrates (9, 10). These carbohydrate synthesis loci are variable yet ubiquitous in A. baumannii (11, 12). Comparison of 12 known capsule structures from A. baumannii with the sequences of their carbohydrate synthesis loci has provided strong evidence that these loci are responsible for capsule synthesis with as many as 77 distinct serotypes identified by molecular serotyping (11). Because of the non-template driven nature of glycan synthesis, the identification and characterization of the glycans themselves are required to confirm the true diversity. This diversity has widespread implications for Acinetobacter biology as the resulting carbohydrate structures are not solely used for capsule biosynthesis but can be incorporated and utilized by other ubiquitous systems, such as O-linked protein glycosylation (13, 14).Although originally thought to be restricted to species such as Campylobacter jejuni (15, 16) and Neisseria meningitidis (17), bacterial protein glycosylation is now recognized as a common phenomenon within numerous pathogens and commensal bacteria (18, 19). Unlike eukaryotic glycosylation where robust and high-throughput technologies now exist to enrich (20–22) and characterize both the glycan and peptide component of glycopeptides (23–25), the diversity (glycan composition and linkage) within bacterial glycosylation systems makes few technologies broadly applicable to all bacterial glycoproteins. Because of this challenge a deeper understanding of the glycan diversity and substrates of glycosylation has been largely unachievable for the majority of known bacterial glycosylation systems. The recent implementation of selective glycopeptide enrichment methods (26, 27) and the use of multiple fragmentation approaches (28, 29) has facilitated identification of an increasing number of glycosylation substrates independent of prior knowledge of the glycan structure (30–33). These developments have facilitated the undertaking of comparative glycosylation studies, revealing glycosylation is widespread in diverse genera and far more diverse then initially thought. For example, Nothaft et al. were able to show N-linked glycosylation was widespread in the Campylobacter genus and that two broad groupings of the N-glycans existed (34).During the initial characterization of A. baumannii O-linked glycosylation the use of selective enrichment of glycopeptides followed by mass spectrometry analysis with multiple fragmentation technologies was found to be an effective means to identify multiple glycosylated substrates in the strain ATCC 17978 (14). Interestingly in this strain, the glycan utilized for protein modification was identical to a single subunit of the capsule (13) and the loss of either protein glycosylation or glycan synthesis lead to decreases in biofilm formation and virulence (13, 14). Because of the diversity in the capsule carbohydrate synthesis loci and the ubiquitous distribution of the PglL O-oligosaccharyltransferase required for protein glycosylation, we hypothesized that the glycan variability might be also extended to O-linked glycosylation. This diversity, although common in surface carbohydrates such as the lipopolysaccharide of numerous Gram-negative pathogens (35), has only recently been observed within bacterial proteins glycosylation system that are typically conserved within species (36) and loosely across genus (34, 37).In this study, we explored the diversity within the O-linked protein glycosylation systems of Acinetobacter species. Our analysis complements the recent in silico studies of A. baumannii showing extensive glycan diversity exists in the carbohydrate synthesis loci (11, 12). Employing global strategies for the analysis of glycosylation, we experimentally demonstrate that the variation in O-glycan structure extends beyond the genetic diversity predicted by the carbohydrate loci alone and targets proteins of similar properties and identity. Using this knowledge, we developed a targeted approach for the detection of protein glycosylation, enabling streamlined analysis of glycosylation within a range of genetic backgrounds. We determined that; O-linked glycosylation is widespread in clinically relevant Acinetobacter species; inter- and intra-strain heterogeneity exist within glycan structures; glycan diversity, although extensive results in the generation of glycans with similar properties and that the utilization of a single glycan for capsule and O-linked glycosylation is a general feature of A. baumannii but may not be a general characteristic of all Acinetobacter species such as A. baylyi.     

Nuclear Dynamics during Germination,Conidiation, and Hyphal Fusion of Fusarium oxysporum

In many fungal pathogens, infection is initiated by conidial germination. Subsequent stages involve germ tube elongation, conidiation, and vegetative hyphal fusion (anastomosis). Here, we used live-cell fluorescence to study the dynamics of green fluorescent protein (GFP)- and cherry fluorescent protein (ChFP)-labeled nuclei in the plant pathogen Fusarium oxysporum. Hyphae of F. oxysporum have uninucleated cells and exhibit an acropetal nuclear pedigree, where only the nucleus in the apical compartment is mitotically active. In contrast, conidiation follows a basopetal pattern, whereby mononucleated microconidia are generated by repeated mitotic cycles of the subapical nucleus in the phialide, followed by septation and cell abscission. Vegetative hyphal fusion is preceded by directed growth of the fusion hypha toward the receptor hypha and followed by a series of postfusion nuclear events, including mitosis of the apical nucleus of the fusion hypha, migration of a daughter nucleus into the receptor hypha, and degradation of the resident nucleus. These previously unreported patterns of nuclear dynamics in F. oxysporum could be intimately related to its pathogenic lifestyle.Fusarium oxysporum is a soilborne pathogen that causes substantial losses in a wide variety of crops (12) and has been reported as an emerging human pathogen (36, 38). Similar to other fungal pathogens (18), the early stages of interaction between F. oxysporum and the host are crucial for the outcome of infection (11). Key processes occurring during these initial stages include spore germination, adhesion to the host surface, establishment of hyphal networks through vegetative hyphal fusion, differentiation of infection hyphae, and penetration of the host (53). Surprisingly, very little is known about the cytology of basic processes, such as spore germination and hyphal development, which play key roles during infection by F. oxysporum.F. oxysporum produces three types of asexual spores: microconidia, macroconidia, and chlamydospores (9, 26). Germination usually represents the first step in the colonization of a new environment, including the host. Once dormancy is broken, spores undergo a defined set of morphogenetic changes that lead to the establishment of a polarized growth axis and the emergence of one or multiple germ tubes (reviewed by d''Enfert and Hardham [10, 19]). In certain fungi, such as Aspergillus nidulans, germ tube emergence and septum formation are subject to precise spatial controls and are tightly coordinated with nuclear division (20, 22, 34, 42, 54). In contrast, in spores from other filamentous fungi, such as macroconidia of Fusarium graminearum, nuclear division is not required for the emergence of germ tubes (21, 48). During hyphal growth, multinucleate fungi display distinct mitotic patterns, such as asynchronous nuclear division in Neurospora crassa and Ashbya gossypii (15, 16, 29, 30, 33, 49), parasynchronous in A. nidulans (7, 15, 23, 46), and synchronous in Ceratocystis fagacearum (1, 15).Vegetative hyphal fusion, or anastomosis, is a common developmental process during the life cycle of filamentous fungi that is thought to serve important functions in intrahyphal communication, nutrient transport, and colony homeostasis (41). F. oxysporum undergoes anastomosis (8, 25, 32, 40), and although this process is not strictly required for plant infection, it appears to contribute to efficient colonization of the root surface (39).The aim of this study was to explore nuclear dynamics during different developmental stages of F. oxysporum that are of key relevance during the establishment of infection. They include germination of microconidia, vegetative hyphal development, and conidiation, as well as vegetative hyphal fusion during colony establishment. Fusion PCR-mediated gene targeting (55) was used to C-terminally label histone H1 in F. oxysporum (FoH1) with either green fluorescent protein (GFP) or the cherry variant (ChFP), allowing us to perform, for the first time, live-cell analysis of nuclear dynamics in this species. Our study revealed distinct patterns of nuclear divisions in F. oxysporum. Moreover, we report, for the first time in an ascomycete, that hyphal fusion initiates a series of nuclear events, including mitosis in the fusing hypha and nuclear migration into the receptor hypha, followed by degradation of the resident nucleus.     

Yeast Cell Adhesion Molecules Have Functional Amyloid-Forming Sequences

The occurrence of highly conserved amyloid-forming sequences in Candida albicans Als proteins (H. N. Otoo et al., Eukaryot. Cell 7:776–782, 2008) led us to search for similar sequences in other adhesins from C. albicans and Saccharomyces cerevisiae. The β-aggregation predictor TANGO found highly β-aggregation-prone sequences in almost all yeast adhesins. These sequences had an unusual amino acid composition: 77% of their residues were β-branched aliphatic amino acids Ile, Thr, and Val, which is more than 4-fold greater than their prevalence in the S. cerevisiae proteome. High β-aggregation potential peptides from S. cerevisiae Flo1p and C. albicans Eap1p rapidly formed insoluble amyloids, as determined by Congo red absorbance, thioflavin T fluorescence, and fiber morphology. As examples of the amyloid-forming ability of the native proteins, soluble glycosylphosphatidylinositol (GPI)-less fragments of C. albicans Als5p and S. cerevisiae Muc1p also formed amyloids within a few days under native conditions at nM concentrations. There was also evidence of amyloid formation in vivo: the surfaces of cells expressing wall-bound Als1p, Als5p, Muc1p, or Flo1p were birefringent and bound the fluorescent amyloid-reporting dye thioflavin T. Both of these properties increased upon aggregation of the cells. In addition, amyloid binding dyes strongly inhibited aggregation and flocculation. The results imply that amyloid formation is an intrinsic property of yeast cell adhesion proteins from many gene families and that amyloid formation is an important component of cellular aggregation mediated by these proteins.Protein amyloids are characteristic of pathological conditions, including neurodegenerative diseases (4, 11, 17, 38). These protein aggregates can also occur naturally in adhesive bacterial curli (3), melanosomes (14), condensed peptide hormone arrays (24), as regulatory prions in yeast (2, 5), and fungal hydrophobins, which are nonantigenic coats to some fungi (1, 33, 39). Nevertheless, such natural occurrences are relatively few, considering the negative free energy for amyloid formation (28).We have recently discovered that there are amyloid-forming sequences in the cell surface Als adhesins of Candida albicans. Cells that express these adhesins aggregate readily, and the aggregation has amyloid-like properties, including protein conformational shifting, surface birefringence, and ability to bind the amyloid-active dyes Congo red and amino-naphthalene sulfonic acid (ANS) (29). A five- to seven-residue sequence in Als1p, Als3p, and Als5p has extremely high potential for formation of β-aggregates, according to the protein state prediction program TANGO (13, 27, 31). Such β-aggregates include amyloids, which are ordered structures with paracrystalline regions of stacked parallel β-strands that are perpendicular to the long axis of micrometer-long fibrils. The strands are stabilized by interaction of identical sequences from many protein molecules (31, 32). Where TANGO analyses have shown that specific sequences have β-aggregate potentials greater than 20%, an insoluble β-aggregate state is likely to form. These β-aggregates nucleate formation of amyloids if the proteins can associate to form fibers (13, 27, 31). Sequences in the conserved 127-residue T region of Als1p, Als3p, and Als5p have β-aggregation potentials of >90% (27). An oligopeptide with this sequence, as well as 412- and 645-residue fragments of Als5p formed authentic amyloids, as determined by characteristic dye binding and fiber morphology. The amyloid-forming sequences were rich in the β-branched amino acids Thr, Val, and Ile. This amino acid composition is unusual among proteins in general, but is common in the Thr-rich mid-piece domains of yeast adhesins.Yeasts display many cell-wall-bound adhesins that mediate colonial and biofilm interactions as well as host-pathogen binding (9, 21, 41). Such adhesins have a common mosaic structure. In general, the adhesins have N-terminal globular binding domains (often immunoglobulin-like or lectin-like), Thr-rich mid-piece sequences including tandem repeats, and 300- to 800-residue heavily glycosylated Ser and Thr-rich “stalk” domains near the C-terminal domain that extend the active regions from the surface of the wall. The adhesins are covalently cross-linked to wall polysaccharides through modified glycosylphosphatidylinositol (GPI) anchors and/or glycosyl esters of glutamic acid (9, 18).Because the yeast adhesins share this common modular domain structure, we searched among known and putative yeast adhesins for sequences with high β-aggregation potential. We have found that many of these proteins share amyloid-forming sequences and amyloid-like behavior on activation.     

Using in Vivo Biotinylated Ubiquitin to Describe a Mitotic Exit Ubiquitome from Human Cells

Mitotic division requires highly regulated morphological and biochemical changes to the cell. Upon commitment to exit mitosis, cells begin to remove mitotic regulators in a temporally and spatially controlled manner to bring about the changes that reestablish interphase. Ubiquitin-dependent pathways target these regulators to generate polyubiquitin-tagged substrates for degradation by the 26S proteasome. However, the lack of cell-based assays to investigate in vivo ubiquitination limits our knowledge of the identity of substrates of ubiquitin-mediated regulation in mitosis. Here we report an in vivo ubiquitin tagging system used in human cells that allows efficient purification of ubiquitin conjugates from synchronized cell populations. Coupling purification with mass spectrometry, we have identified a series of mitotic regulators targeted for polyubiquitination in mitotic exit. We show that some are new substrates of the anaphase-promoting complex/cyclosome and validate KIFC1 and RacGAP1/Cyk4 as two such targets involved respectively in timely mitotic spindle disassembly and cell spreading. We conclude that in vivo biotin tagging of ubiquitin can provide valuable information about the role of ubiquitin-mediated regulation in processes required for rebuilding interphase cells.Ubiquitination has emerged as a major post-translational modification determining the fate of cellular proteins. One of these fates is proteolysis, whereby the assembly of polyubiquitin chains creates signatures on target proteins that specify delivery to the 26S proteasome for proteolytic destruction. Targeted proteolysis is critical to the control of cell division. For example, the universally conserved mechanism of mitotic exit depends upon rapid proteolysis of mitotic cyclins and securins to drive the transition from mitosis to interphase. This transition is under surveillance by the spindle assembly checkpoint (SAC),¹ which controls the activity of a multi-subunit ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C) (1, 2).Much of the known specificity in the ubiquitin-proteasome system (UPS) is mediated at the level of substrate targeting by ubiquitin ligase (E3) enzymes, of which there are more than 600 in human cells. Given these facts, it is perhaps surprising that the APC/C is almost the only known engineer of the protein landscape after anaphase onset, targeting mitotic regulators for destruction with high temporal specificity (2–4). Some roles for nondegradative ubiquitination in regulating the localization of mitotic kinases Aurora B and Plk1 have been described (5–9), and a growing list of reported ubiquitin interactors can modulate ubiquitin-dependent events during mitosis (10). However, the majority of ubiquitination events that have so far been described as occurring at the transition from mitosis to interphase are APC/C-dependent.Two co-activator subunits, Cdc20 and Cdh1, play vital roles in APC/C-dependent substrate recognition (11) by recognizing two widely characterized degrons, the D-box and the KEN motif (12, 13). Computational approaches that have been used to calculate the total number of APC/C substrates from the prevalence of degrons in the human proteome estimate that there are between 100 and 200 substrates (14), and experiments using in vitro ubiquitination of protein arrays have given rise to estimates in the same range (15). Most of the mitotic regulators targeted by the APC/C during mitotic exit in human cells have been identified via in vitro degradation assays or ubiquitination assays on in vitro–expressed pools of substrates (15–18). These approaches have identified several important substrates, but in the absence of in vivo parameters they may not identify substrates whose targeting depends on post-translational modifications or substrates that are only recognized in vivo as components of higher-order complexes. Not all substrates identified in this way have been validated as polyubiquitinated proteins in vivo. Multiple recent proteomic studies have identified large numbers of in vivo ubiquitin-modified sites from yeast (19–21) and human cells (22–29). None of these studies have used synchronized cell populations to provide information on the timing or regulation of substrate ubiquitination.We reasoned that a better view of ubiquitin-mediated processes that regulate mitotic exit would come from identifying proteins that are ubiquitinated in vivo during mitotic exit. With this goal in mind we adopted a system for in vivo tagging of ubiquitin chains with biotin, previously used to identify ubiquitin-conjugated proteins from the Drosophila neural system (30), and applied it to a human cell line (U2OS) that can be tightly synchronized at mitosis. In contrast to several recent studies that employed antibodies specific to the diGly-Lys remnant that marks ubiquitination sites following trypsin digestion (19, 25), an in vivo ubiquitin tagging strategy allows direct validation of candidate ubiquitinated proteins (whether mono- or polyubiquitinated) through immunoblotting of samples. Moreover, in contrast to other methods for affinity tagging of ubiquitin, or affinity purification via ubiquitin-binding domains, the use of the biotin tag enables purification under highly denaturing conditions for stringent isolation of ubiquitin-conjugated material from higher eukaryotes. His₆-tagged ubiquitin is also available for use under denaturing conditions, but it is not generally useful in higher eukaryotic cells, where a high frequency of proteins containing multiple histidine residues confounds the specificity of nickel-affinity pulldowns (as discussed in detail in Ref. 30). Therefore, in this paper we describe the reproducible identification and validation of mitoticphase-specific polyubiquitinated proteins via the in vivo biotinylation of ubiquitin. A large number of polyubiquitinated proteins that we identified are specific to mitotic exit, when the APC/C is active, and we expect that many of them are substrates for the APC/C. We formally identified KIFC1/HSET and Cyk4/RACGAP1 as targets of APC/C-dependent ubiquitin-mediated proteolysis after anaphase onset and investigated the role of their ubiquitination in the regulation of mitotic exit. Cell cycle phase-specific information on protein ubiquitination and the generation of ubiquitinated protein networks provides a framework for further investigation of ubiquitin-controlled processes occurring during the rebuilding of interphase cells.     

The Antiretroviral Lectin Cyanovirin-N Targets Well-Known and Novel Targets on the Surface of Entamoeba histolytica Trophozoites

Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, has a truncated Asn-linked glycan (N-glycan) precursor composed of seven sugars (Man₅GlcNAc₂). Here, we show that glycoproteins with unmodified N-glycans are aggregated and capped on the surface of E. histolytica trophozoites by the antiretroviral lectin cyanovirin-N and then replenished from large intracellular pools. Cyanovirin-N cocaps the Gal/GalNAc adherence lectin, as well as glycoproteins containing O-phosphodiester-linked glycans recognized by an anti-proteophosphoglycan monoclonal antibody. Cyanovirin-N inhibits phagocytosis by E. histolytica trophozoites of mucin-coated beads, a surrogate assay for amebic virulence. For technical reasons, we used the plant lectin concanavalin A rather than cyanovirin-N to enrich secreted and membrane proteins for mass spectrometric identification. E. histolytica glycoproteins with occupied N-glycan sites include Gal/GalNAc lectins, proteases, and 17 previously hypothetical proteins. The latter glycoproteins, as well as 50 previously hypothetical proteins enriched by concanavalin A, may be vaccine targets as they are abundant and unique. In summary, the antiretroviral lectin cyanovirin-N binds to well-known and novel targets on the surface of E. histolytica that are rapidly replenished from large intracellular pools.Entamoeba histolytica causes amebic dysentery and liver abscess in the developing world (10, 20, 29). We are interested in E. histolytica glycoproteins containing Asn-linked glycans (N-glycans) for numerous reasons. E. histolytica makes an N-glycan precursor that contains 7 sugars (Man₅GlcNAc₂-PP-dolichol) rather than 14 sugars (Glc₃Man₉GlcNAc₂-PP-dolichol) made by most animals, plants, and fungi (21, 31, 44). E. histolytica N-glycans are used for quality control of glycoprotein folding in the endoplasmic reticulum (ER) lumen, and there is positive selection for sites of N-linked glycosylation in secreted and membrane proteins of E. histolytica (5, 11, 53).Unprocessed Man₅GlcNAc₂, by far the most abundant E. histolytica N-glycan, is present on the plasma membrane and vesicular membranes (31). The antiretroviral lectin cyanovirin-N, which is specific for α-1,2-linked mannose present on unprocessed N-glycans, binds E. histolytica N-glycans and forms aggregates or caps on the surface of E. histolytica trophozoites (1, 25, 31, 44, 45). E. histolytica glycoproteins are also capped by the plant lectin concanavalin A (ConA), which has a broader carbohydrate specificity (mannose and glucose) than cyanovirin-N (3, 16, 18, 19). Heavy subunits of the Gal/GalNAc lectin, the most important E. histolytica vaccine candidate, have 7 to 10 potential sites for N-linked glycosylation (32, 39, 43). Inhibition of N-glycan synthesis results in Gal/GalNAc lectins that are unable to bind to sugars on host epithelial cells.Carbohydrates appear to be an important target on the surface of E. histolytica as anti-proteophosphoglycan (PPG) monoclonal antibodies bind to O-phosphodiester-linked glycans and protect animal models from amebic infection (6, 33, 35, 40, 48). Lectin affinity columns are a powerful method for enriching unique parasite glycoproteins that may be identified by mass spectrometry (MS) of tryptic fragments (17, 55). For example, we recently used the plant lectin wheat germ agglutinin to dramatically enrich glycoproteins with short N-glycans of Giardia (42).The goal of the present studies was to explore further the interaction of the antiretroviral lectin cyanovirin-N with E. histolytica trophozoites in vitro. Questions asked included the following: Are E. histolytica glycoproteins with N-glycans replenished on the plasma membrane after capping with cyanovirin-N? What is the effect of cyanovirin-N capping on other amebic virulence factors and/or vaccine candidates (e.g., the Gal/GalNAc lectin and PPG)? Is capping by cyanovirin-N mediated by actin, as described for capping by the Gal/GalNAc lectin and ConA? What is the effect of the cyanovirin-N on amebic phagocytosis of mucin-coated beads, a surrogate assay for virulence? Which trophozoite glycoproteins are potential targets of cyanovirin-N (identified by mass spectrometry of lectin-enriched E. histolytica proteins)? Are any of them potential vaccine candidates?     

A Novel Protein Kinase Localized to Lipid Droplets Is Required for Droplet Biogenesis in Trypanosomes

Ubiquitous among eukaryotes, lipid droplets are organelles that function to coordinate intracellular lipid homeostasis. Their morphology and abundance is affected by numerous genes, many of which are involved in lipid metabolism. In this report we identify a Trypanosoma brucei protein kinase, LDK, and demonstrate its localization to the periphery of lipid droplets. Association with lipid droplets was abrogated when the hydrophobic domain of LDK was deleted, supporting a model in which the hydrophobic domain is associated with or inserted into the membrane monolayer of the organelle. RNA interference knockdown of LDK modestly affected the growth of mammalian bloodstream-stage parasites but did not affect the growth of insect (procyclic)-stage parasites. However, the abundance of lipid droplets dramatically decreased in both cases. This loss was dominant over treatment with myriocin or growth in delipidated serum, both of which induce lipid body biogenesis. Growth in delipidated serum also increased LDK autophosphorylation activity. Thus, LDK is required for the biogenesis or maintenance of lipid droplets and is one of the few protein kinases specifically and predominantly associated with an intracellular organelle.Trypanosoma brucei is a single-celled eukaryotic pathogen responsible for human African trypanosomiasis (also known as African sleeping sickness) and nagana in domestic animals. More than 50,000 cases of human disease occur yearly, with over 70 million people at risk. No vaccine exists, and chemotherapy is difficult to administer and prone to pathogen resistance. As T. brucei transits between the mammalian bloodstream and the tsetse fly vector during its life cycle, the organism encounters and adapts to profoundly different environmental conditions. The parasite undergoes dramatic changes in both energy (7, 51) and lipid biosynthesis and metabolism (39, 47, 49) as it shifts between these environments.Protein kinases function in numerous regulatory aspects of the cell, including control of the cell cycle and morphology, responses to stress, and transmission of signals from the extracellular environment or between compartments of the cell. As is the case in other eukaryotes, protein kinases, particularly those associated with membranes, are expected to play pivotal roles in the cell''s ability to sense and appropriately respond to its environment. Trypanosoma brucei possesses over 170 protein kinases (16, 44). Most of these can be assigned to the standard groups of protein kinases based on sequence similarity within the kinase domain. However, sequence similarities with kinases from more well-studied organisms are rarely strong enough to allow one-to-one orthologous relationships to be determined (44), and even those which appear orthologous by sequence have sometimes shown functional divergence (46). Hence, an understanding of the roles of specific protein kinases of trypanosomatids requires an individualized assessment. The initial genome analysis of the trypanosomatids (16) showed a lack of receptor tyrosine kinases, but nine T. brucei predicted serine/threonine kinases were annotated as possessing transmembrane domains. One of these was recently shown to be strategically located at a key interface between the host and parasite: the flagellar pocket (38). This eukaryotic translation initiation factor 2α (eIF2α) family kinase was postulated to play a sensory role in monitoring protein transport.Only a very small number of protein kinases of various organisms have been observed to localize to the membranes of intracellular organelles, most of them to the endoplasmic reticulum (ER) (14, 27, 50). Lipid droplets (also known as lipid bodies, adiposomes, or oil bodies in plants) are thought to arise from the ER, although the routes of protein localization to them are not well understood. They are increasingly recognized as legitimate organelles due to their dynamic roles in energy metabolism (40), lipid trafficking (41), and protection against toxic effects of nonesterified lipids and sterols (18). Studies also suggest that they function as potential protein storage depots (12) and in antigen presentation (10). Although recent efforts to expand the lipid droplet proteome have resulted in a vastly increased and in many cases surprising catalogue of potentially associated proteins (3, 5, 11, 12, 23, 37), relatively little is known as to how these structures form and are regulated within the cell.We examine here a novel T. brucei protein kinase with a predicted transmembrane domain. Surprisingly, this protein is localized intracellularly in association with lipid droplets. RNAi-mediated knockdown of this newly identified kinase, dubbed LDK (for lipid droplet kinase), reveals a role in the formation or maintenance of lipid droplets in both mammalian bloodstream-form (BF) and insect procyclic-form (PF) stages of the parasite life cycle.     

Suggestive Evidence for Darwinian Selection against Asparagine-Linked Glycans of Plasmodium falciparum and Toxoplasma gondii

We are interested in asparagine-linked glycans (N-glycans) of Plasmodium falciparum and Toxoplasma gondii, because their N-glycan structures have been controversial and because we hypothesize that there might be selection against N-glycans in nucleus-encoded proteins that must pass through the endoplasmic reticulum (ER) prior to threading into the apicoplast. In support of our hypothesis, we observed the following. First, in protists with apicoplasts, there is extensive secondary loss of Alg enzymes that make lipid-linked precursors to N-glycans. Theileria makes no N-glycans, and Plasmodium makes a severely truncated N-glycan precursor composed of one or two GlcNAc residues. Second, secreted proteins of Toxoplasma, which uses its own 10-sugar precursor (Glc₃Man₅GlcNAc₂) and the host 14-sugar precursor (Glc₃Man₉GlcNAc₂) to make N-glycans, have very few sites for N glycosylation, and there is additional selection against N-glycan sites in its apicoplast-targeted proteins. Third, while the GlcNAc-binding Griffonia simplicifolia lectin II labels ER, rhoptries, and surface of plasmodia, there is no apicoplast labeling. Similarly, the antiretroviral lectin cyanovirin-N, which binds to N-glycans of Toxoplasma, labels ER and rhoptries, but there is no apicoplast labeling. We conclude that possible selection against N-glycans in protists with apicoplasts occurs by eliminating N-glycans (Theileria), reducing their length (Plasmodium), or reducing the number of N-glycan sites (Toxoplasma). In addition, occupation of N-glycan sites is markedly reduced in apicoplast proteins versus some secretory proteins in both Plasmodium and Toxoplasma.Animals, fungi, and plants synthesize Asn-linked glycans (N-glycans) by means of a lipid-linked precursor containing 14 sugars (dolichol-PP-Glc₃Man₉GlcNAc₂) (26). Recently we used bioinformatics and experimental methods to show that numerous protists are missing sets of glycosyltransferases (Alg1 to Alg14) and so make truncated N-glycan precursors containing 0 to 11 sugars (46). For example, Entamoeba histolytica, which causes dysentery, makes N-glycan precursors that contain seven sugars (Man₅GlcNAc₂) (33). Giardia lamblia, a cause of diarrhea, makes N-glycan precursors that contain just GlcNAc₂ (41). N-glycan precursors may be identified by metabolic labeling with radiolabeled mannose (Entamoeba) or glucosamine (Giardia) (46). Unprocessed N-glycans of each protist may be recognized by wheat germ agglutinin 1 (WGA-1) (GlcNAc₂ of Giardia) or by the antiretroviral lectin cyanovirin-N (Man₅GlcNAc₂ of Entamoeba) (2, 33, 41).N-glycans are transferred from lipid-linked precursors to sequons (Asn-Xaa-Ser or Asn-Xaa-Thr, where Xaa cannot be Pro) on nascent peptides by an oligosaccharyltransferase (OST) (28). For the most part, transfer of N-glycans by the OST is during translocation, although there are human and Trypanosoma OSTs that transfer N-glycans after translocation (34, 45).N-glycan-dependent quality control (QC) systems for protein folding and endoplasmic reticulum (ER)-associated degradation (ERAD), which are present in most eukaryotes, are missing from Giardia and a few other protists that make truncated N-glycans (5, 26, 53). There is positive Darwinian selection for sequons (sites of N-glycans) that contain Thr in secreted and membrane proteins of organisms that have N-glycan-dependent QC (12). This selection occurs for the most part by an increased probability that Asn and Thr will be present in sequons rather than elsewhere in secreted and membrane proteins. In contrast, there is no selection on sequons that contain Ser, and there is no selection on sequons in the secreted proteins of organisms that lack N-glycan-dependent QC.For numerous reasons, we are interested in the N-glycans of Plasmodium falciparum and Toxoplasma gondii, which cause severe malaria and disseminated infections, respectively.(i) There has been controversy for a long time as to whether Plasmodium makes N-glycans. While some investigators identified a 14-sugar Plasmodium N-glycan resembling that of the human host (29), others identified no N-glycans (6, 22).(ii) There is also controversy concerning whether the N-glycans of Toxoplasma, after removal of Glc by glucosidases in the ER lumen, contain either 7 sugars (Man₅GlcNAc₂), like Entamoeba (32, 33), or 11 sugars (Man₉GlcNAc₂), like the human host (16, 19, 26). If it is Man₅GlcNAc₂, then Toxoplasma uses the dolichol-PP-linked glycan predicted by its set of Alg enzymes (32, 46). If it is Man₉GlcNAc₂, then Toxoplasma uses the dolichol-PP-linked glycan of the host cell (16, 19, 26).(iii) Both Plasmodium and Toxoplasma are missing proteins involved in N-glycan-dependent QC of protein folding (5).(iv) We hypothesize that there may be negative selection against N-glycans in Plasmodium and Toxoplasma, because the N-glycans added in the ER lumen during translocation will likely interfere with threading of nucleus-encoded apicoplast proteins into a nonphotosynthetic, chloroplast-derived organelle called the apicoplast (21, 35, 37, 48, 52, 54). Nucleus-encoded apicoplast proteins have a bipartite signal at the N terminus, which targets proteins first to the lumen of the ER and second to lumen of the apicoplast. This bipartite signal has been used in transformed plasmodia where green fluorescent protein (GFP) is targeted to the apicoplast with the bipartite signal of the acyl carrier protein (ACP_leader-GFP), to the secretory system with the signal sequence only (ACP_signal-GFP), and to the cytosol with the organelle-targeting transit peptide only (ACP_transit-GFP) (55). Similar constructs have been used to characterize signals that target nucleus-encoded proteins of Toxoplasma to the apicoplast (11, 25).Here we use a combination of bioinformatic, biochemical, and morphological methods to characterize the N-glycans of Plasmodium and Toxoplasma and to test our hypothesis that there is negative selection against N-glycans in protists with apicoplasts.