No abstract

Phosphatidylinositol 3-kinase Vps34 complexes regulate intracellular membrane trafficking in endocytic sorting, cytokinesis, and autophagy. We present the 4.4 angstrom crystal structure of the 385-kilodalton endosomal complex II (PIK3C3-CII), consisting of Vps34, Vps15 (p150), Vps30/Atg6 (Beclin 1), and Vps38 (UVRAG). The subunits form a Y-shaped complex, centered on the Vps34 C2 domain. Vps34 and Vps15 intertwine in one arm, where the Vps15 kinase domain engages the Vps34 activation loop to regulate its activity. Vps30 and Vps38 form the other arm that brackets the Vps15/Vps34 heterodimer, suggesting a path for complex assembly. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal conformational changes accompanying membrane binding and identify a Vps30 loop that is critical for the ability of complex II to phosphorylate giant liposomes on which complex I is inactive.

The root-hair pattern of Arabidopsis is determined through a regulatory circuit composed of transcription factor genes. The homeobox gene GLABRA2 (GL2) has been considered a key component, acting farthest downstream in this regulation. GL2 modified to include a transactivating function caused epidermal cells to develop ectopic root hairs or root hair-like structures. With this system, the phospholipase Dzeta1 gene (AtPLDzeta1) was identified as a direct target of GL2. Inducible expression of AtPLDzeta1 promoted ectopic root-hair initiation. We conclude that GL2 exerts its regulatory effect on root-hair development through modulation of phospholipid signaling.

Plant cells frequently undergo endoreduplication, a process in which chromosomal DNA is successively duplicated in the absence of mitosis. It has been proposed that endoreduplication is regulated at its entry by mitotic cyclin-dependent kinase activity. However, the regulatory mechanisms for its termination remain unclear, although plants tightly control the ploidy level in each cell type. In the process of searching for regulatory factors of endoreduplication, the promoter of an Arabidopsis thaliana cyclin A gene, CYCA2;3, was revealed to be active in developing trichomes during the termination period of endoreduplication as well as in proliferating tissues. Taking advantage of the situation that plants encode highly redundant cyclin A genes, we were able to perform functional dissection of CYCA2;3 using null mutant alleles. Null mutations of CYCA2;3 semidominantly promoted endocycles and increased the ploidy levels achieved in mature organs, but they did not significantly affect the proportion of cells that underwent endoreduplication. Consistent with this result, expression of the CYCA2;3-green fluorescent protein fusion protein restrained endocycles in a dose-dependent manner. Moreover, a mutation in the destruction box of CYCA2;3 stabilized the fusion protein in the nuclei and enhanced the restraint. We conclude that CYCA2;3 negatively regulates endocycles and acts as a key regulator of ploidy levels in Arabidopsis endoreduplication.

The integral membrane protein Atg9 is delivered to the autophagosome in yeast and mammalian cells. We find that Atg9 does not originate from mitochondria and cannot reach the autophagosome directly from the ER. Instead, pairwise combinations of mutations in Golgi-endosomal traffic components cause defects in Atg9 delivery during starvation.

The Arabidopsis thaliana GLABRA2 (GL2) gene encodes a transcription factor involved in the cell differentiation of various epidermal tissues. During root hair pattern formation, GL2 suppresses root hair development in non-hair cells, acting as a node between the gene regulatory networks for cell fate determination and cell differentiation. Despite the importance of GL2 function, its molecular basis remains obscure because the GL2 target genes leading to the network for cell differentiation are unknown. We identified five basic helix-loop-helix (bHLH) transcription factor genes (ROOT HAIR DEFECTIVE6, and LRL2) as GL2 direct targets using transcriptional and posttranslational induction systems. Chromatin immunoprecipitation analysis confirmed GL2 binding to upstream regions of these genes in planta. Reporter gene analyses showed that these genes are expressed in various stages of root hair development and are suppressed by GL2 in non-hair cells. GL2 promoter-driven GFP fusions of LRL1 and LRL2, but not those of the other bHLH proteins, conferred root hair development on non-hair cells. These results indicate that GL2 directly suppresses bHLH genes with diverse functions in root hair development.

The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor–Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor–Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor–Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended ‘railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit.

BackgroundHOX genes encode a family of homeodomain-containing transcription factors involved in the determination of cell fate and identity during embryonic development. They also behave as oncogenes in some malignancies.ResultsIn this study, we found high expression of the HOXD9 gene transcript in glioma cell lines and human glioma tissues by quantitative real-time PCR. Using immunohistochemistry, we observed HOXD9 protein expression in human brain tumor tissues, including astrocytomas and glioblastomas. To investigate the role of HOXD9 in gliomas, we silenced its expression in the glioma cell line U87 using HOXD9-specific siRNA, and observed decreased cell proliferation, cell cycle arrest, and induction of apoptosis. It was suggested that HOXD9 contributes to both cell proliferation and/or cell survival. The HOXD9 gene was highly expressed in a side population (SP) of SK-MG-1 cells that was previously identified as an enriched-cell fraction of glioma cancer stem-like cells. HOXD9 siRNA treatment of SK-MG-1 SP cells resulted in reduced cell proliferation. Finally, we cultured human glioma cancer stem cells (GCSCs) from patient specimens found with high expression of HOXD9 in GCSCs compared with normal astrocyte cells and neural stem/progenitor cells (NSPCs).ConclusionsOur results suggest that HOXD9 may be a novel marker of GCSCs and cell proliferation and/or survival factor in gliomas and glioma cancer stem-like cells, and a potential therapeutic target.