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The pathological accumulation of cholesterol is a signature feature of Niemann-Pick type C (NPC) disease, in which excessive lipid levels induce Purkinje cell death in the cerebellum. NPC1 encodes a lysosomal cholesterol-binding protein, and mutations in NPC1 drive cholesterol accumulation in late endosomes and lysosomes (LE/Ls). However, the fundamental role of NPC proteins in LE/L cholesterol transport remains unclear. Here, we demonstrate that NPC1 mutations impair the projection of cholesterol-containing membrane tubules from the surface of LE/Ls. A proteomic survey of purified LE/Ls identified StARD9 as a novel lysosomal kinesin responsible for LE/L tubulation. StARD9 contains an N-terminal kinesin domain, a C-terminal StART domain, and a dileucine signal shared with other lysosome-associated membrane proteins. Depletion of StARD9 disrupts LE/L tubulation, paralyzes bidirectional LE/L motility and induces accumulation of cholesterol in LE/Ls. Finally, a novel StARD9 knock-out mouse recapitulates the progressive loss of Purkinje cells in the cerebellum. Together, these studies identify StARD9 as a microtubule motor protein responsible for LE/L tubulation and provide support for a novel model of LE/L cholesterol transport that becomes impaired in NPC disease. [ABSTRACT FROM AUTHOR]

Cytoplasmic dynein functions at several sites during mitosis; however, the basis of targeting to each site remains unclear. Tandem mass spectrometry analysis of mitotic dynein revealed a phosphorylation site in the dynein intermediate chains (ICs) that mediates binding to kinetochores. IC phosphorylation directs binding to zw10 rather than dynactin, and this interaction is needed for kinetochore dynein localization. Phosphodynein associates with kinetochores from nuclear envelope breakdown to metaphase, but bioriented microtubule (MT) attachment and chromosome alignment induce IC dephosphorylation. IC dephosphorylation stimulates binding to dynactin and poleward streaming. MT depolymerization, release of kinetochore tension, and a PPI-[gamma] mutant each inhibited IC dephosphorylation, leading to the retention of phosphodynein at kinetochores and reduced poleward streaming. The depletion of kinetochore dynactin by moderate levels of p50(dynamitin) expression disrupted the ability of dynein to remove checkpoint proteins by streaming at metaphase but not other aspects of kinetochore dynein activity. Together, these results suggest a new model for localization of kinetochore dynein and the contribution of kinetochore dynactin.

Kinesin II is a heterotrimeric plus end-directed microtubule motor responsible for the anterograde movement of organelles in various cell types. Despite substantial literature concerning the types of organelles that kinesin II transports, the question of how this motor associates with cargo organelles remains unanswered. To address this question, we have used Xenopus laevis melanophores as a model system. Through analysis of kinesin II-mediated melanosome motility, we have determined that the dynactin complex, known as an anchor for cytoplasmic dynein, also links kinesin II to organelles. Biochemical data demonstrates that the putative cargo-binding subunit of Xenopus kinesin II, Xenopus kinesin II-associated protein (XKAP), binds directly to the p[150.sup.Glued] subunit of dynactin. This interaction occurs through aa 530-793 of XKAP and aa 600-811 of p[150.sup.Glued]. These results reveal that dynactin is required for transport activity of microtubule motors of opposite polarity, cytoplasmic dynein and kinesin II, and may provide a new mechanism to coordinate their activities.

A subset of microtubule-associated proteins, including cytoplasmic linker protein (CLIP)-170, dynactin, EB1, adenomatous polyposis coli, cytoplasmic dynein, CLASPs, and LIS-1, has been shown recently to target to the plus ends of microtubules. The mechanisms and functions of this binding specificity are not understood, although a role in encouraging microtubule elongation has been proposed. To extend previous work on the role of dynactin in organelle transport, we analyzed [p150.sup.Glued] by live-cell imaging. Time-lapse analysis of [p150.sup.Glued] revealed targeting to the plus ends of growing microtubules, requiring the N[H.sub.2]-terminal cytoskeleton-associated protein-glycine rich domain, but not EB1 or CLIP-170. Effectors of protein kinase A modulated microtubule binding and suggested [p150.sup.Glued] phosphorylation as a factor in plus-end binding specificity. Using a phosphosensitive monoclonal antibody, we mapped the site of [p150.sup.Glued] phosphorylation to Ser-19. In vivo and in vitro analysis of phosphorylation site mutants revealed that [p150.sup.Glued] phosphorylation mediates dynamic binding to microtubules. To address the function of dynamic binding, we imaged GFP-[p150.sup.Glued] during the dynein-dependent transport of Golgi membranes. Live-cell analysis revealed a transient interaction between Golgi membranes and GFP-[p150.sup.Glued]-labeled microtubules just prior to transport, implicating microtubules and dynactin in a searchcapture mechanism for minus-end-directed organelles.

Results reveal that BZAP45 protein possesses a decapeptide as a part of the putative leucine-zipper protein with an ATP or GTP nucleotide binding fold. Data indicate that purified BZAP45 interacts with site II cell cycle control element indirectly in cooperation with H4 promoter.

The purification of the histone nuclear factor M has been achieved and it is found to be a protein with molecular mass of 48K which is the same as interferon regulatory factor 2 (IRF-2). The transcription of the human histone gene reaches a maximum limit during the early S phase. IRF-2 has an oncogenic action and there is a link between IRF-2 and a gene that is linked to DNA replication and the transition from G1 to S phase of cell cycle.

Previous studies have implicated the dynactin complex as a receptor or adaptor for cytoplasmic dynein on the surface of cargo. This interaction is mediated by the cytoplasmic dynein intermediate chains (ICs) and [p150.sup.Glued]. To identify regulatory mechanisms that control the binding of cytoplasmic dynein to cargo, we mapped phosphorylation sites on the ICs and [p150.sup.Glued], and determined the impact of phosphorylation on protein function. A [p150.sup.Glued] phosphorylation site was identified in the [NH.sub.2]-terminal 'head' domain through mapping of the epitope of a phospho-sensitive antibody. This phosphorylation site resides near the microtubule-binding domain of [p150.sup.Glued], and fits the consensus for cAMP-dependent PKA. Using GFP-fusions, we visualized site-directed mutants in living cells and determined that [p150.sup.Glued] phosphorylation regulates the dynamic targeting of dynactin to microtubule plus-ends. To test IC phosphorylation as a regulatory mechanism, we analyzed cytoplasmic dynein by 2-D gel analysis and detected two IC variants, one of which was eliminated by phosphatase treatment. To measure the effect of this IC phosphorylation on the IC-[p150.sup.Glued] interaction, we performed overlay assays and observed that [p150.sup.Glued] bound dephosphorylated but not phosphorylated ICs. To map the site of IC phosphorylation, we subjected the purified cytoplasmic dynein ICs to mass spectrometry. A single phosphorylated tryptic fragment was identified corresponding to the [p150.sup.Glued]-binding domain of IC-2C. Fragmentation and retention time analysis were used to map the phosphorylation site to serine 84. Site-directed mutants designed to mimic the dephosphorylated (S84A) or phosphorylated (S84D) IC disrupted both in vitro and in vivo IC phosphorylation. S84A mutants bound [p150.sup.Glued] in vitro and overexpression perturbed transport of dynein-dependent membranes. The S84D mutant diminished [p50.sup.Glued] binding in vitro and did not disrupt dynein-mediated transport when expressed in vivo. These findings represent the first evidence that IC phosphorylation plays a fundamental role in the binding of cytoplasmic dynein to dynactin and cargo. (Supported by AHA grant 3009N.)

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Kinetochore dynein has been implicated in microtubule capture, correcting inappropriate microtubule attachments, chromosome movement, and checkpoint silencing. It remains unclear how dynein coordinates this diverse set of functions. Phosphorylation is responsible for some dynein heterogeneity (Whyte, J., Bader, J. R., Tauhata, S. B., Raycroft, M., Hornick, J., Pfister, K. K., Lane, W. S., Chan, G. K., Hinchcliffe, E, H., Vaughan, P.S., and Vaughan, K. T. (2008) J. Cell Biol. 183, 819- 834), and phosphorylated and dephosphorylated forms of dynein coexist at prometaphase kinetochores. In this study, we measured the impact of inhibiting polo-like kinase 1 (Plk1) on both dynein populations. Phosphorylated dynein was ablated at kinetochores after inhibiting Plk1 with a small molecule inhibitor (5-Cyano-7-nitro-2-(benzothiazolo-N-oxide)-carboxamide) or chemical genetic approaches. The total complement of kinetochore dynein was also reduced but not eliminated, reflecting the presence of some dephosphorylated dynein after Plk1 inhibition. Although Plk1 inhibition had a profound effect on dynein, kinetochore populations of dynactin, spindly, and zw10 were not reduced. Plk1-independent dynein was reduced after p150Glued depletion, consistent with the binding of dephosphorylated dynein to dynactin. Plk1 phosphorylated dynein intermediate chains at Thr-89 in vitro and generated the phospho-Thr-89 phosphoepitope on recombinant dynein intermediate chains. Finally, inhibition of Plk1 induced defects in microtubule capture and persistent microtubule attachment, suggesting a role for phosphorylated dynein in these functions during prometaphase. These findings suggest that Plk1 is a dynein kinase required for recruitment of phosphorylated dynein to kinetochores. [ABSTRACT FROM AUTHOR]

Aurora B (AurB) is a mitotic kinase responsible for multiple aspects of mitotic progression, including assembly of the outer kinetochore. Cytoplasmic dynein is an abundant kinetochore protein whose recruitment to kinetochores requires phosphorylation. To assess whether AurB regulates recruitment of dynein to kinetochores, we inhibited AurB using ZM447439 or a kinase-dead AurB construct. Inhibition of AurB reduced accumulation of dynein at kinetochores substantially; however, this reflected a loss of dynein-associated proteins rather than a defect in dynein phosphorylation. We determined that AurB inhibition affected recruitment of the ROD, ZW10, zwilch (RZZ) complex to kinetochores but not zwint-1 or more-proximal kinetochore proteins. AurB phosphorylated zwint-1 but not ZW10 in vitro, and three novel phosphorylation sites were identified by tandem mass spectrometry analysis. Expression of a triple-Ala zwint-1 mutant blocked kinetochore assembly of RZZ-dependent proteins and induced defects in chromosome movement during prometaphase. Expression of a triple-Glu zwint-1 mutant rendered cells resistant to AurB inhibition during prometaphase. However, cells expressing the triple-Glu mutant failed to satisfy the spindle assembly checkpoint (SAC) at metaphase because poleward streaming of dynein/dynactin/RZZ was inhibited. These studies identify zwint-1 as a novel AurB substrate required for kinetochore assembly and for proper SAC silencing at metaphase.

Cytoplasmic dynein is the major minus-end directed microtubule-based motor in eukaryotic cells. It is composed of a number of different subunits including three light chain families: Tctex1, LC8, and roadblock. The incorporation of the roadblock light chains into the cytoplasmic dynein complex had not been determined. There are two roadblock genes in mammals, ROBL-1 and ROBL-2. We find that both members of the roadblock family bind directly to all of the intermediate chain isoforms of mammalian cytoplasmic dynein. This was determined with three complementary approaches. A yeast two-hybrid assay demonstrated that both roadblock light chains interact with intermediate chain isoforms from the IC74-1 and IC74-2 genes in vivo. This was confirmed in vitro with both a solid phase blot overlay assay and a solution-binding assay. The roadblock-binding domain on the intermediate chain was mapped to an approximately 72 residue region. The binding domain is downstream of each of the two alternative splice sites in the intermediate chains. This location is consistent with the finding that both roadblock-1 and roadblock-2 show no binding specificity for a single IC74-1 or IC74-2 intermediate chain isoform. In addition, this roadblock-binding domain is significantly downstream from both the Tctex1- and LC8-binding sites, supporting the hypothesis that multiple light chain family members can bind to the same intermediate chain.