Abstract. The subcellular distribution of microtubules containing acetylated a-tubulin in mammalian cells in culture was analyzed with 6-11B-l, a monoclonal antibody specific for acetylated a-tubulin. Cultures of 3T3, HeLa, and PtK2 cells were grown on coverslips and observed by immunofluorescence microscopy after double-staining by 6-11B-1 and B-5-1-2, a monoclonal antibody specific for all ¢t-tubulins. The antibody 6-11B-1 binds to primary cilia, centrioles, mitotic spindles, midbodies, and to subsets of cytoplasmic microtubules in 3T3 and HeLa cells, but not in PtK2 cells. These observations confirm that the acetylation of a-tubulin is a modification occurring in different microtubule structures and in a variety of eukaryotic cells. Some features of the acetylation of cytoplasmic microtubules of mammalian cells are also described here. First, acetylated a-tubulin is present in microtubules that, under depolymerizing conditions, are more stable than the majority of cytoplasmic microtubules. In addition to the specific microtubule frameworks already mentioned, cytoplasmic microtubules resistant to nocodazole or colchicine, but not cold-resistant microtubules, contain more acetylated a-tubulin than the rest of cellular microtubules. Second, the ¢t-tubulin in all cytoplasmic microtubules of 3T3 and HeLa cells becomes acetylated in the presence of taxol, a drug that stabilizes microtubules. Third, acetylation and deacetylation of cytoplasmic microtubules are reversible in cells released from exposure to 0°C or antimitotic drugs. Fourth, the epitope recognized by the antibody 6-11B-1 is not absolutely necessary for cell growth and division. This epitope is absent in PtK2 cells. The acetylation of et-tubulin could regulate the presence of microtubules in specific intracellular spaces by selective stabilization.

Innate immune system activation is a critical step in the initiation of an effective adaptive immune response; therefore, activation of a class of innate pathogen receptors called pattern recognition receptors (PRR) is a central feature of many adjuvant systems. It has recently been shown that one member of an intracellular PRR, the NLRP3 inflammasome, is activated by a number of classical adjuvants including aluminum hydroxide and saponins [1,2]. Inflammasome activation in vitro requires signaling of both the Toll-like receptor (TLR) and NLRP3 in antigen-presenting cells. Here we present a class of nanomaterials endowed with these two signals for rapid optimization of vaccine design. We constructed this system using a simple approach that incorporates lipopolysaccharides (LPS) onto the surface of nanoparticles constructed from a biocompatible polyester, poly (lactic-coglycolic acid) (PLGA), loaded with antigen. We demonstrate that LPS-modified particles are preferentially internalized by dendritic cells compared to uncoated nanoparticles and the system, when administered to mice, elicits potent humoral and cellular immunity against a model antigen, ovalbumin. Wild type macrophages pulsed with LPS-modified nanoparticles resulted in production of the proinflammatory cytokine IL-1β consistent with inflammasome activation. In comparison, NLRP3-deficient and caspase-1-deficient macrophages showed negligible production of IL-1β. Furthermore, when endocytosis and lysosomal destabilization were inhibited, inflammasome activity was diminished, supporting the notion that nanoparticles rupture lysosomal compartments and behave as 'danger signals' [3]. The generality of this vaccination approach is tested by encapsulation of a recombinant West Nile envelope protein and demonstrated by protection against a murine model of West Nile encephalitis. The design of such an antigen delivery mechanism with the ability to

An acetylation site of Chlamydomonas axonemal a-tubulins was identified near, or within, the binding site of 6-11B-1i a monoclonal antibody specific for posttranslationally acetylated cr-tubulins. In a first approach, axonemal proteins were hydrolyzed by formic acid, cyanogen bromide, or chymotrypsin and analyzed with immunoblots. The smallest ax-tubulin peptide retained on nitrocellulose and containing antibody-binding site(s) was found to span amino acids 37-138 (a37-138). A smaller antibody-binding peptide, identified as a25-50, was obtained by complete digestion of a-tubulin with chymotrypsin. This fragment was purified by reversed-phase HPLC and assayed by its ability to bind 6-11B-1 in solution. Determination of the amino acid sequences of a37-138 and a25-50 showed that residue 40 in axonemal a-tubulin is eN acetyllysine. A sequence very similar to Chlamydomonas a25-50 is found in the majority of a-tubulins analyzed so far. However, the corresponding region is markedly divergent in some a-tubulin isoforms from chicken, Drosophila, and yeast.Progress in the biochemistry and molecular genetics of tubulins and proteins that interact with the microtubules has been reviewed in the proceedings of a recent conference (1). Since microtubules participate in a wide variety of cellular functions, much attention has been devoted to analyzing possible mechanisms of microtubule differentiation. For example, a greater resistance to depolymerizing drugs distinguishes specific microtubule structures as well as subsets of cytoplasmic microtubules. Such differential stability of microtubules has been correlated, in some cases, with the occurrence of C-terminal detyrosination (2) and posttranslational acetylation (3, 4) of assembled at-tubulin. However, the process of microtubule stabilization remains unknown.The presence of modified subunits in microtubules can be monitored by antibodies specific for acetylated (3-9) or detyrosinated (10) a-tubulin. As a step toward understanding the role of a-tubulin acetylation in microtubule stabilization, we set out to determine possible acetylation sites by identifying the binding site(s) of a monoclonal antibody, 6-11B-1, specific for posttranslationally acetylated a-tubulins.MATERIALS AND METHODS Preparation of Chlamydomonas Axonemes. Chlamydomonas 137+ cells were grown and axonemes were isolated essentially as described (11). Axonemes were immediately solubilized in 1% NaDodSO4/15 mM 2-mercaptoethanol.Hybridomas. Hybridomas (5) were grown in HT medium as described (12). 6-11B-1 IgGs were purified from serum-free culture medium (HB102, New England Nuclear) by protein A-Sepharose affinity chromatography and 125I-labeled by the chloramine-T method. After labeling, the 6-11B-1 IgG concentration was 50 ,g/ml and the specific radioactivity was 1.3 x 106 cpm/ul. Gel Electrophoresis, Transfer to Nitrocellulose, and AntiBody Binding. These were essentially as described (5), except that 7.5-15% acrylamide Neville (13) gradient gels were used.Electroelutions. Electroelutions were perfo...

We have used anti-peptide antibodies raised against highly conserved regions of the kinesin motor domain to identify kinesin-related proteins in the fission yeast Schizosaccharomyces pombe. Here we report the identification of a new kinesin-related protein, which we have named pkl1. Sequence homology and domain organization place pkl1 in the Kar3/ncd subfamily of kinesin-related proteins. Bacterially expressed pkl1 fusion proteins display microtubule-stimulated ATPase activity, nucleotide-sensitive binding, and bundling of microtubules. Immunofluorescence studies with affinity-purified antibodies indicate that the pkl1 protein localizes to the nucleus and the mitotic spindle. Pkl1 null mutants are viable but have increased sensitivity to microtubule-disrupting drugs. Disruption of pkl1+ suppresses mutations in another kinesin-related protein, cut7, which is known to act in the spindle. Overexpression of pkl1 to very high levels causes a similar phenotype to that seen in cut7 mutants: V-shaped and star-shaped microtubule structures are observed, which we interpret to be spindles with unseparated spindle poles. These observations suggest that pkl1 and cut7 provide opposing forces in the spindle. We propose that pkl1 functions as a microtubule-dependent motor that is involved in microtubule organization in the mitotic spindle.

West Nile virus, a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specific antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.