P150 Subunit Research Articles

Proteomic Analysis of the Mammalian Katanin Family of Microtubule-severing Enzymes Defines Katanin p80 subunit B-like 1 (KATNBL1) as a Regulator of Mammalian Katanin Microtubule-severing

The Katanin family of microtubule-severing enzymes is critical for remodeling microtubule-based structures that influence cell division, motility, morphogenesis and signaling. Katanin is composed of a catalytic p60 subunit (A subunit, KATNA1) and a regulatory p80 subunit (B subunit, KATNB1). The mammalian genome also encodes two additional A-like subunits (KATNAL1 and KATNAL2) and one additional B-like subunit (KATNBL1) that have remained poorly characterized. To better understand the factors and mechanisms controlling mammalian microtubule-severing, we have taken a mass proteomic approach to define the protein interaction module for each mammalian Katanin subunit and to generate the mammalian Katanin family interaction network (Katan-ome). Further, we have analyzed the function of the KATNBL1 subunit and determined that it associates with KATNA1 and KATNAL1, it localizes to the spindle poles only during mitosis and it regulates Katanin A subunit microtubule-severing activity in vitro. Interestingly, during interphase, KATNBL1 is sequestered in the nucleus through an N-terminal nuclear localization signal. Finally KATNB1 was able to compete the interaction of KATNBL1 with KATNA1 and KATNAL1. These data indicate that KATNBL1 functions as a regulator of Katanin A subunit microtubule-severing activity during mitosis and that it likely coordinates with KATNB1 to perform this function.

Tyrosine Phosphorylation of Caspase-8 Abrogates Its Apoptotic Activity and Promotes Activation of c-Src.

Src family tyrosine kinases (SFKs) phosphorylate caspase-8A at tyrosine (Y) 397 resulting in suppression of apoptosis. In addition, the phosphorylation of caspase-8A at other sites including Y465 has been implicated in the regulation of caspase-8 activity. However, the functional consequences of these modifications on caspase-8 processing/activity have not been elucidated. Moreover, various Src substrates are known to act as potent Src regulators, but no such role has been explored for caspase-8. We asked whether the newly identified caspase-8 phosphorylation sites might regulate caspase-8 activation and conversely, whether caspase-8 phosphorylation might affect Src activity. Here we show that Src phosphorylates caspase-8A at multiple tyrosine sites; of these, we have focused on Y397 within the linker region and Y465 within the p12 subunit of caspase-8A. We show that phosphomimetic mutation of caspase-8A at Y465 prevents its cleavage and the subsequent activation of caspase-3 and suppresses apoptosis. Furthermore, simultaneous phosphomimetic mutation of caspase-8A at Y397 and Y465 promotes the phosphorylation of c-Src at Y416 and increases c-Src activity. Finally, we demonstrate that caspase-8 activity prevents its own tyrosine phosphorylation by Src. Together these data reveal that dual phosphorylation converts caspase-8 from a pro-apoptotic to a pro-survival mediator. Specifically, tyrosine phosphorylation by Src renders caspase-8 uncleavable and thereby inactive, and at the same time converts it to a Src activator. This novel dynamic interplay between Src and caspase-8 likely acts as a potent signal-integrating switch directing the cell towards apoptosis or survival.

A tale of two α-tubulin tails.

Post‐translational modifications of tubulin, such as the removal of the C‐terminal tyrosine of α‐tubulin, have long been proposed to influence the ability of microtubule motors to walk along the microtubule surface. This hypothesis has now been tested for cytoplasmic dynein‐1 (dynein), revealing that active dynein–dynactin–adaptor complexes prefer to start moving on tyrosinated microtubules. This choice is governed by the p150 subunit of dynactin. Once moving, however, dynein is not choosy about whether the microtubule is tyrosinated or not.

Uracil DNA glycosylase interacts with the p32 subunit of the replication protein A complex to modulate HIV-1 reverse transcription for optimal virus dissemination.

BackgroundThrough incorporation into virus particles, the HIV-1 Vpr protein participates in the early steps of the virus life cycle by influencing the reverse transcription process. We previously showed that this positive impact on reverse transcription was related to Vpr binding to the uracil DNA glycosylase 2 enzyme (UNG2), leading to enhancement of virus infectivity in established CD4-positive cell lines via a nonenzymatic mechanism.ResultsWe report here that Vpr can form a trimolecular complex with UNG2 and the p32 subunit (RPA32) of the replication protein A (RPA) complex and we explore how these cellular proteins can influence virus replication and dissemination in the primary target cells of HIV-1, which express low levels of both proteins. Virus infectivity and replication in peripheral blood mononuclear cells and monocyte-derived macrophages (MDMs), as well as the efficiency of the viral DNA synthesis, were significantly reduced when viruses were produced from cells depleted of endogenous UNG2 or RPA32. Moreover, viruses produced in macrophages failed to replicate efficiently in UNG2- and RPA32-depleted T lymphocytes. Reciprocally, viruses produced in UNG2-depleted T cells did not replicate efficiently in MDMs confirming the positive role of UNG2 for virus dissemination.ConclusionsOur data show the positive effect of UNG2 and RPA32 on the reverse transcription process leading to optimal virus replication and dissemination between the primary target cells of HIV-1.

Tyrosination of α-tubulin controls the initiation of processive dynein-dynactin motility.

Post-translational modifications (PTMs) of α/β-tubulin are believed to regulate interactions with microtubule-binding proteins. A well-characterized PTM involves in the removal and re-ligation of the C-terminal tyrosine on α-tubulin, but the purpose of this tyrosination-detyrosination cycle remains elusive. Here, we examined the processive motility of mammalian dynein complexed with dynactin and BicD2 (DDB) on tyrosinated versus detyrosinated microtubules. Motility was decreased ~fourfold on detyrosinated microtubules, constituting the largest effect of a tubulin PTM on motor function observed to date. This preference is mediated by dynactin's microtubule-binding p150 subunit rather than dynein itself. Interestingly, on a bipartite microtubule consisting of tyrosinated and detyrosinated segments, DDB molecules that initiated movement on tyrosinated tubulin continued moving into the segment composed of detyrosinated tubulin. This result indicates that the α-tubulin tyrosine facilitates initial motor-tubulin encounters, but is not needed for subsequent motility. Our results reveal a strong effect of the C-terminal α-tubulin tyrosine on dynein-dynactin motility and suggest that the tubulin tyrosination cycle could modulate the initiation of dynein-driven motility in cells.

SLK/LOSK kinase regulates cell motility independently of microtubule organization and Golgi polarization.

Cell motility is an essential complex process that requires actin and microtubule cytoskeleton reorganization and polarization. Such extensive rearrangement is closely related to cell polarization as a whole. The serine/threonine kinase SLK/LOSK is a potential regulator of cell motility, as it phosphorylates a series of cytoskeleton-bound proteins that collectively participate in the remodeling of migratory cell architecture. In this work, we report that SLK/LOSK is an indispensable regulator of cell locomotion that primarily acts through the small GTPase RhoA and the dynactin subunit p150(Glued). Both RhoA and dynactin affect cytoskeleton organization, polarization, and general cell locomotory activity to various extents. However, it seems that these events are independent of each other. Thus, SLK/LOSK kinase effectively functions as a switch that links all of the processes underlying cell motility to provide robust directional movement.

Optimizing a Male Reproductive Aging Mouse Model by D-Galactose Injection.

The d-galactose (d-gal)-injected animal model, which is typically established by administering consecutive subcutaneous d-gal injections to animals for approximately six or eight weeks, has been frequently used for aging research. In addition, this animal model has been demonstrated to accelerate aging in the brain, kidneys, liver and blood cells. However, studies on aging in male reproductive organs that have used this animal model remain few. Therefore, the current study aimed to optimize a model of male reproductive aging by administering d-gal injections to male mice and to determine the possible mechanism expediting senescence processes during spermatogenesis. In this study, C57Bl/6 mice were randomized into five groups (each containing 8–10 mice according to the daily intraperitoneal injection of vehicle control or 100 or 200 mg/kg dosages of d-gal for a period of six or eight weeks). First, mice subjected to d-gal injections for six or eight weeks demonstrated considerably decreased superoxide dismutase activity in the serum and testis lysates compared to those in the control group. The lipid peroxidation in testis also increased in the d-gal-injected groups. Furthermore, the d-gal-injected groups exhibited a decreased ratio of testis weight/body weight and sperm count compared to the control group. The percentages of both immotile sperm and abnormal sperm increased considerably in the d-gal-injected groups compared to those of the control group. To determine the genes influenced by the d-gal injection during murine spermatogenesis, a c-DNA microarray was conducted to compare testicular RNA samples between the treated groups and the control group. The d-gal-injected groups exhibited RNA transcripts of nine spermatogenesis-related genes (Cycl2, Hk1, Pltp, Utp3, Cabyr, Zpbp2, Speer2, Csnka2ip and Katnb1) that were up- or down-regulated by at least two-fold compared to the control group. Several of these genes are critical for forming sperm-head morphologies or maintaining nuclear integration (e.g., cylicin, basic protein of sperm head cytoskeleton 2 (Cylc2), casein kinase 2, alpha prime interacting protein (Csnka2ip) and katanin p80 (WD40-containing) subunit B1 (Katnb1)). These results indicate that d-gal-injected mice are suitable for investigating male reproductive aging.

Dataset for the NMR structure of the intrinsically disordered acidic region of XPC bound to the PH domain of TFIIH p62.

The global genome nucleotide excision repair factor XPC firstly detects DNA lesions and then recruits a ten-subunit complex TFIIH through binding to the subunit p62 to unwind the damaged DNA for excision repair. This data article contains detailed nuclear magnetic resonance (NMR) restraints (nuclear Overhauser enhancement (NOE)-derived distance restraints, dihedral angle restraints, and hydrogen bond restraints) used for the structure determination of the complex formed between the intrinsically disordered acidic region of XPC and the pleckstrin homology (PH) domain of TFIIH p62, related to the recent work entitled “Structural insight into the mechanism of TFIIH recognition by the acidic string of the nucleotide excision repair factor XPC.” [1].

A Proteomic Study of Memory After Imprinting in the Domestic Chick.

The intermediate and medial mesopallium (IMM) of the domestic chick forebrain has previously been shown to be a memory system for visual imprinting. Learning-related changes occur in certain plasma membrane and mitochondrial proteins in the IMM. Two-dimensional gel electrophoresis/mass spectrometry has been employed to identify more comprehensively learning-related expression of proteins in the membrane-mitochondrial fraction of the IMM 24 h after training. We inquired whether amounts of these proteins in the IMM and a control region (posterior pole of the nidopallium, PPN) are correlated with a behavioral estimate of memory for the imprinting stimulus. Learning-related increases in amounts of the following proteins were found in the left IMM, but not the right IMM or the left or right PPN: (i) membrane cognin; (ii) a protein resembling the P32 subunit of splicing factor SF2; (iii) voltage-dependent anionic channel-1; (iv) dynamin-1; (v) heterogeneous nuclear ribonucleoprotein A2/B1. Learning-related increases in some transcription factors involved in mitochondrial biogenesis were also found, without significant change in mitochondrial DNA copy number. The results indicate that the molecular processes involved in learning and memory underlying imprinting include protein stabilization, increased mRNA trafficking, synaptic vesicle recycling, and specific changes in the mitochondrial proteome.

Mutations in the Dynein1 Complex are Permissible for Basal Body Migration in Photoreceptors but Alter Rab6 Localization.

The photoreceptor outer segment is a specialized primary cilium, and anchoring of the basal body at the apical membrane is required for outer segment formation. We hypothesized that basal body localization and outer segment formation would require the microtubule motor dynein 1 and analyzed the zebrafish cannonball and mike oko mutants, which carry mutations in the heavy chain subunit of cytoplasmic dynein 1 (dync1h1) and the p150(Glued) subunit of Dynactin (dctn1a). The distribution of Rab6, a player in the post-Golgi trafficking of rhodopsin, was also examined. Basal body docking was unaffected in both mutants, but Rab6 expression was reduced. The results suggest that dynein 1 is dispensable for basal body docking but that outer segment defects may be due to defects in post-Golgi trafficking.

ID: 107: Inhibition of type I IFN induction by Sandfly fever Sicilian virus virulence factor NSs

Phleboviruses are a group of emerging viruses with a wide spectrum of virulence: For example, Rift Valley Fever virus (RVFV) is highly pathogenic, whereas Sandfly fever Sicilian virus (SFSV) causes intermediate pathogenicity. The major virulence factor is the non-structural protein NSs, a suppressor of the type I interferon (IFN) system. While the NSs protein of highly pathogenic RVFV inhibits global host cell transcription via sequestration and degradation of the TFIIH subunits p44 and p62, respectively, the mode of action of the NSs protein of intermediately pathogenic SFSV has remained elusive. We therefore aimed to characterize the IFN-inhibitory function employed by the NSs of SFSV. Using mass spectrometry, our group has identified multiple candidate host interactors, among them interferon regulatory factor 3 (IRF3). Indeed, co-immunoprecipitation confirmed the interaction of SFSV NSs with IRF3, but not with other IRF family members like IRF2, IRF5 and IRF9. Furthermore, SFSV NSs specifically abrogated IRF3 activation and thus IFN- β promoter activity, but not general host cell transcription. Hence, SFSV encodes a NSs protein that efficiently inhibits the induction of type I IFN. Different to the highly pathogenic RVFV, which induces a general host transcription shutoff, however, SFSV specifically targets IRF3-dependent IFN promoter activation. Although RVFV and SFSV are highly related, their NSs proteins display remarkably diverse strategies of counteracting the type I IFN system. Possibly, the quality of NSs-host factor interactions correlates with the virulence levels of phleboviruses.

Structural Insight into the Mechanism of TFIIH Recognition by the Acidic String of the Nucleotide Excision Repair Factor XPC

In global genome repair (GGR), XPC detects damaged nucleotides and recruits TFIIH complex. The small acidic region of XPC binds to the pleckstrin homology (PH) domain of TFIIH subunit p62; however, the recognition mechanism remains elusive. Here, we use nuclear magnetic resonance to present the tertiary structure of XPC bound to the PH domain. The XPC acidic region forms a long string stabilized by insertion of Trp133 and Val136 into two separate hollows of the PH domain, coupled with extensive electrostatic contacts. Analysis of several XPC mutants revealed that particularly Trp133 is essential for binding to the PH domain. In cell lines stably expressing mutant XPC, alanine substitution at Trp133 or Trp133/Val136 compromised UV resistance, recruitment of TFIIH to DNA damage, and removal of UV-induced photoproducts from genomic DNA. These findings show how TFIIH complex is recruited by XPC to damaged DNA, advancing our understanding of the early stage of GGR.

Grabbing the genome by the NADs.

The regions of the genome that interact frequently with the nucleolus have been termed nucleolar-associated domains (NADs). Deep sequencing and DNA-fluorescence in situ hybridization (FISH) experiments have revealed that these domains are enriched for repetitive elements, regions of the inactive X chromosome (Xi), and several RNA polymerase III-transcribed genes. NADs are often marked by chromatin modifications characteristic of heterochromatin, including H3K27me3, H3K9me3, and H4K20me3, and artificial targeting of genes to this area is correlated with reduced expression. It has therefore been hypothesized that NAD localization to the nucleolar periphery contributes to the establishment and/or maintenance of heterochromatic silencing. Recently published studies from several multicellular eukaryotes have begun to reveal the trans-acting factors involved in NAD localization, including the insulator protein CCCTC-binding factor (CTCF), chromatin assembly factor (CAF)-1 subunit p150, several nucleolar proteins, and two long non-coding RNAs (lncRNAs). The mechanisms by which these factors coordinate with one another in regulating NAD localization and/or silencing are still unknown. This review will summarize recently published studies, discuss where additional research is required, and speculate about the mechanistic and functional implications of genome organization around the nucleolus.

Real-time and simultaneous monitoring of the phosphorylation and enhanced interaction of p53 and XPC acidic domains with the TFIIH p62 subunit.

Posttranslational modifications have critical roles in diverse biological processes through interactions. Tumor-suppressor protein p53 and nucleotide excision repair factor XPC each contain an acidic region, termed the acidic transactivation domain (TAD) and acidic fragment (AF), respectively, that binds to the pleckstrin homology (PH) domain of the p62 subunit of the transcription factor TFIIH. Human p53-TAD contains seven serine and two threonine residues, all of which can be phosphorylated. Similarly, XPC-AF contains six serine and two threonine residues, of which Thr117, Ser122 and Ser129 have been reported as phosphorylation sites in vivo, although their phosphorylation roles are unknown. Phosphorylation of Ser46 and Thr55 of p53-TAD increases its binding ability; however, the role of XPC-AF phosphorylation remains elusive. Here we describe a system for real-time and simultaneous monitoring of the phosphorylation and p62-PH affinity of p53-TAD and XPC-AF using nuclear magnetic resonance (NMR) spectroscopy. Unexpectedly, among seven reported kinases that presumably phosphorylate Ser46 and/or Thr55 of p53-TAD, only two specific and high-efficiency enzymes were identified: JNK2α2 for Ser46 and GRK5 for Thr55. During interaction with p62-PH, four different affinity complexes resulting from various phosphorylation states of p53-TAD by the kinases were identified. The kinetics of the site-specific phosphorylation reaction of p53-TAD and its affinity for p62-PH were monitored in real-time using the NMR system. Isothermic calorimetry showed that phosphorylation of Ser129 of XPC-AF increases binding to p62-PH. Although CK2 was predicted to phosphorylate Ser122, Ser129 and Ser140 from its sequence context, it specifically and efficiently phosphorylated only Ser129. Simultaneous monitoring of the phosphorylation and augmentation in p62-PH binding identified a key residue of p62-PH for contacting phosphorylated Ser129. In summary, we have established an NMR system for real-time and simultaneous monitoring of site-specific phosphorylation and enhancement of affinity between phosphorylation domains and their target. The system is also applicable to other posttranslational modifications.

Characterizing the effect of Bortezomib on Rift Valley Fever Virus multiplication

Rift Valley Fever Virus (RVFV) belongs to the family Bunyaviridae and is a known cause of epizootics and epidemics in Africa and the Middle East. With no FDA approved therapeutics available to treat RVFV infection, understanding the interactions between the virus and the infected host is crucial to developing novel therapeutic strategies. Here, we investigated the requirement of the ubiquitin–proteasome system (UPS) for the establishment of a productive RVFV infection. It was previously shown that the UPS plays a central role in RVFV multiplication involving degradation of PKR and p62 subunit of TFIIH. Using the FDA-approved proteasome inhibitor Bortezomib, we observed robust inhibition of intracellular and extracellular viral loads. Bortezomib treatment did not affect the nuclear/cytoplasmic distribution of the non-structural S-segment protein (NSs); however, the ability of NSs to form nuclear filaments was abolished as a result of Bortezomib treatment. In silico ubiquitination prediction analysis predicted that known NSs interactors (SAP30, YY1, and mSin3A) have multiple putative ubiquitination sites, while NSs itself was not predicted to be ubiquitinated. Immunoprecipitation studies indicated a decrease in interaction between SAP30 – NSs, and mSin3A – NSs in the context of Bortezomib treatment. This decrease in association between SAP30 – NSs also correlated with a decrease in the ubiquitination status of SAP30 with Bortezomib treatment. Bortezomib treatment, however, resulted in increased ubiquitination of mSin3A, suggesting that Bortezomib dynamically affects the ubiquitination status of host proteins that interact with NSs. Finally, we observed that expression of interferon beta (IFN-β) was increased in Bortezomib treated cells which indicated that the cellular antiviral mechanism was revived as a result of treatment and may contribute to control of viral multiplication.

Initiation and termination of DNA replication during S phase in relation to cyclins D1, E and A, p21WAF1, Cdt1 and the p12 subunit of DNA polymerase δ revealed in individual cells by cytometry.

During our recent studies on mechanism of the regulation of human DNA polymerase δ in preparation for DNA replication or repair, multiparameter imaging cytometry as exemplified by laser scanning cytometry (LSC) has been used to assess changes in expression of the following nuclear proteins associated with initiation of DNA replication: cyclin A, PCNA, Ki-67, p21(WAF1), DNA replication factor Cdt1 and the smallest subunit of DNA polymerase δ, p12. In the present review, rather than focusing on Pol δ, we emphasize the application of LSC in these studies and outline possibilities offered by the concurrent differential analysis of DNA replication in conjunction with expression of the nuclear proteins. A more extensive analysis of the data on a correlation between rates of EdU incorporation, likely reporting DNA replication, and expression of these proteins, is presently provided. New data, specifically on the expression of cyclin D1 and cyclin E with respect to EdU incorporation as well as on a relationship between expression of cyclin A vs. p21(WAF1) and Ki-67 vs. Cdt1, are also reported. Of particular interest is the observation that this approach makes it possible to assess the temporal sequence of degradation of cyclin D1, p21(WAF1), Cdt1 and p12, each with respect to initiation of DNA replication and with respect to each other. Also the sequence or reappearance of these proteins in G2 after termination of DNA replication is assessed. The reviewed data provide a more comprehensive presentation of potential markers, whose presence or absence marks the DNA replicating cells. Discussed is also usefulness of these markers as indicators of proliferative activity in cancer tissues that may bear information on tumor progression and have a prognostic value.

A ΩXaV motif in the Rift Valley fever virus NSs protein is essential for degrading p62, forming nuclear filaments and virulence

Rift Valley fever virus (RVFV) is a single-stranded RNA virus capable of inducing fatal hemorrhagic fever in humans. A key component of RVFV virulence is its ability to form nuclear filaments through interactions between the viral nonstructural protein NSs and the host general transcription factor TFIIH. Here, we identify an interaction between a ΩXaV motif in NSs and the p62 subunit of TFIIH. This motif in NSs is similar to ΩXaV motifs found in nucleotide excision repair (NER) factors and transcription factors known to interact with p62. Structural and biophysical studies demonstrate that NSs binds to p62 in a similar manner as these other factors. Functional studies in RVFV-infected cells show that the ΩXaV motif is required for both nuclear filament formation and degradation of p62. Consistent with the fact that the RVFV can be distinguished from other Bunyaviridae-family viruses due to its ability to form nuclear filaments in infected cells, the motif is absent in the NSs proteins of other Bunyaviridae-family viruses. Taken together, our studies demonstrate that p62 binding to NSs through the ΩXaV motif is essential for degrading p62, forming nuclear filaments and enhancing RVFV virulence. In addition, these results show how the RVFV incorporates a simple motif into the NSs protein that enables it to functionally mimic host cell proteins that bind the p62 subunit of TFIIH.

PDIP46 – A Pol δ and PCNA Binding Protein that Stimulates Human Pol δ Activity

PDIP46 (poldip3) is a 46 kDa protein which interacts with human DNA polymerase δ (Pol δ) via the p50 subunit, and whose biochemical effects on Pol δ are unknown. The objectives of this study were to characterize the effects of PDIP46 on Pol δ activity. The interaction sites between the p50 subunit of Pol δ and PDIP46 were mapped. PDIP46 was also found to interact with PCNA, which it does via multiple copies of a novel PCNA binding motif (APIM ‐ AlkB homologue 2 PCNA‐Interacting Motif). Both the p50 and PCNA interaction regions are located in the N‐terminus. PDIP46 potently activates Pol δ activity (ca. 10‐fold with an apparent KD of ca. 34 nM) on singly primed 7 kb ssM13 DNA in an assay which requires highly processive synthesis. Further dissection of the effects of PDIP46 using model oligonucleotide templates showed that it stimulated primer extension and strand displacement reactions in the presence of PCNA. PDIP46 also stimulated primer extension in the absence of PCNA, indicating that it has the ability to exert a direct effect on Pol δ. PDIP46 was associated with Pol δ on DNA as shown by chromatin immunoprecipitation using antibody against the Pol δ p125 subunit. These in vitro studies show that PDIP46 has the biochemical attributes to function as a novel accessory protein for Pol δ that accelerates its activity, and thus is a candidate for a role as a cellular regulator of Pol δ.

Impact of three inactivated bovine viral diarrhoea virus vaccines on bulk milk p80 (NS3) ELISA test results in dairy herds

Bovine viral diarrhoea virus (BVDV) is endemic in many countries and vaccines are used as a component of control and eradication strategies. Surveillance programmes to detect exposure to BVDV often incorporate the use of bulk milk (BM) testing for antibodies against BVDV p80 (NS3), but vaccination can interfere with these results. The aim of this study was to evaluate whether BVDV vaccines would confound BM testing for specific antibodies in a nationally representative group of commercial dairy farms in the Republic of Ireland. A total of 256 commercial dairy herds were included in the statistical analysis. Quarterly BM or serum samples from selected weanling heifers (unvaccinated homeborn youngstock) were assessed by ELISA for antibodies against the BVDV p80 subunit and whole virus. Wilcoxon rank-sum and receiver operating characteristic (ROC) analyses were used to examine differences among groups vaccinated with one of three commercially available inactivated BVDV vaccines. Two of the three vaccines showed evidence of interference with ELISA testing of BM samples. ROC analysis highlighted that one vaccine did not reduce the discriminatory power of the BVDV p80 ELISA for identification of herds with evidence of recent BVDV circulation, when compared with unvaccinated herds; thus, administration of this vaccine would allow uncomplicated interpretation of BM ELISA test results in vaccinated seropositive herds. Seasonal differences in BM antibody results were identified, suggesting that the latter half of lactation is the most suitable time for sampling dairy herds containing predominantly spring calving cows. The results of the present study are likely to prove useful in countries allowing vaccination during or following BVDV eradication, where BM testing is required as part of the surveillance strategy.

A PIK3C3-ankyrin-B-dynactin pathway promotes axonal growth and multiorganelle transport.

Axon growth requires long-range transport of organelles, but how these cargoes recruit their motors and how their traffic is regulated are not fully resolved. In this paper, we identify a new pathway based on the class III PI3-kinase (PIK3C3), ankyrin-B (AnkB), and dynactin, which promotes fast axonal transport of synaptic vesicles, mitochondria, endosomes, and lysosomes. We show that dynactin associates with cargo through AnkB interactions with both the dynactin subunit p62 and phosphatidylinositol 3-phosphate (PtdIns(3)P) lipids generated by PIK3C3. AnkB knockout resulted in shortened axon tracts and marked reduction in membrane association of dynactin and dynein, whereas it did not affect the organization of spectrin-actin axonal rings imaged by 3D-STORM. Loss of AnkB or of its linkages to either p62 or PtdIns(3)P or loss of PIK3C3 all impaired organelle transport and particularly retrograde transport in hippocampal neurons. Our results establish new functional relationships between PIK3C3, dynactin, and AnkB that together promote axonal transport of organelles and are required for normal axon length.