One of the most powerful techniques for attributing functions to genes in uni-and multicellular organisms is comprehensive analysis of mutant traits. In this study, systematic and quantitative analyses of mutant traits are achieved in the budding yeast Saccharomyces cerevisiae by investigating morphological phenotypes. Analysis of fluorescent microscopic images of triple-stained cells makes it possible to treat morphological variations as quantitative traits. Deletion of nearly half of the yeast genes not essential for growth affects these morphological traits. Similar morphological phenotypes are caused by deletions of functionally related genes, enabling a functional assignment of a locus to a specific cellular pathway. The high-dimensional phenotypic analysis of defined yeast mutant strains provides another step toward attributing gene function to all of the genes in the yeast genome.cell morphology ͉ functional genomics ͉ high-dimensional phenotyping ͉ phenome O ne of the ultimate goals of genetics is to reveal relationships between gene function and phenotypic traits. Comprehensive analysis of mutant traits is a very powerful technique for attributing functions to genes in uni-and multicellular organisms. In the budding yeast Saccharomyces cerevisiae, a complete set of mutants, each of which carries a precise deletion of one yeast ORF, has been systematically constructed (1). By using these mutant strains combined with microarray and robot technology, genome-wide analyses of various mutant traits, including general growth rate, fitness under a particular condition, and sensitivity to drugs, has been reported (reviewed in ref. 2).Cell morphology becomes an attractive target for comprehensive analysis, because more powerful methods for fluorescent microscopic imaging analysis in biological research have been emerging after development of high-resolution microscopes and specific fluorescent dyes. Yeast cell morphology reflects various cellular events, including progression through the cell cycle, establishment of cell polarity, and regulation of cell size control. Previous genome-wide studies of yeast morphology were focused on a specific morphology, such as cell size, cell shape, or bud site pattern (3-6), and therefore extracted limited information. Because morphological traits are often judged ''by eye,'' it has remained difficult to obtain quantitative and reproducible results.We recently developed an image-processing system that automatically processes digital cell images of each yeast cell (7,8) to obtain quantitative morphological data of yeast mutant cells. Mannoprotein (as a cell wall component marker), the actin cytoskeleton, and nuclear DNA are specifically stained simultaneously. Cells are then photographed, and fluorescence images are automatically processed. The obtained images of all yeast mutants and data-mining functions are available at our Saccharomyces cerevisiae Morphological Database (SCMD) web site (8,9).In this study, we employ high-dimensional and quantitative phenotyping of yeast muta...

Cyclin-dependent kinase 5 (CDK5) is a unique CDK, the activity of which can be detected in postmitotic neurons. To date, CDK5 purified from mammalian brains has always been associated with a truncated form of the 35-kDa major brain specific activator (p35, also known as nck5a) of CDK5, known as p25. In this study, we report that p35 can be cleaved to p25 both in vitro and in vivo by calpain. In a rat brain extract, p35 was cleaved to p25 by incubation with Ca 2؉ . This cleavage was inhibited by a calpain inhibitor peptide derived from calpastatin and was ablated by separating the p35⅐CDK5 from calpain by centrifugation. The p35 recovered in the pellet after centrifugation could then be cleaved to p25 by purified calpain. Cleavage of p35 was also induced in primary cultured neurons by treatment with a Ca 2؉ ionophore and Ca 2؉ and inhibited by calpain inhibitor I. The cleavage changed the solubility of the CDK5 active complex from the particulate fraction to the soluble fraction but did not affect the histone H1 kinase activity. Increased cleavage was detected in cultured neurons undergoing cell death, suggesting a role of the cleavage in neuronal cell death.Cyclin-dependent kinases (CDKs) 1 are a group of serine/ threonine protein kinases activated by binding to a regulatory subunit cyclin. These kinases are key regulators of the eukaryotic cell cycle progression (1, 2), but only CDK5 appears to function in postmitotic neurons (3). Although the CDK5 protein is expressed at basal levels in most mammalian tissues (4 -7), CDK5 activity has been demonstrated only in brains and developing muscle (6,8), as these are the only tissues that express CDK5 activators (9 -11). The major CDK5 activator in the brain is p35 (also termed nck5a) (9, 10, 12). As a deficiency of CDK5 or p35 results in a reversed layer formation of cortical neurons in the cerebrum or a defect in the formation of the cortical laminar structure in the cerebellum (13, 14), p35⅐CDK5 is thought to play an important role in brain development.Extensive studies of CDK activation in proliferating cells have shown that the activity of CDKs is tightly regulated by the synthesis/degradation of their respective cyclin partners and the phosphorylation/dephosphorylation of their catalytic subunits (1, 2). However, CDK5 does not need to be phosphorylated to regulate kinase activity. Even though the phosphorylation sites for both activation and inhibition of this protein have been conserved (3-5, 15-17), CDK5 can be activated by binding to p35 or the C-terminal fragment of p35 (a 25-kDa regulatory protein known as p25, formed when p35 is nicked between Phe-98 and Ala-99) (9, 10, 12). Recent studies have shown that p35 level is regulated by proteasome degradation (18,19), as the level of cyclins in proliferating cells has been shown (1, 2). Cyclins are periodically synthesized and degraded during the cell cycle (1, 2), but the neuronal activities involved in the synthesis and degradation of p35 are unknown.CDK5 appears to be involved in the migration or positioning...

It has been thought that clathrin-mediated endocytosis is regulated by phosphorylation and dephosphorylation of many endocytic proteins, including amphiphysin I and dynamin I. Here, we show that Cdk5/p35-dependent cophosphorylation of amphiphysin I and dynamin I plays a critical role in such processes. Cdk5 inhibitors enhanced the electric stimulation–induced endocytosis in hippocampal neurons, and the endocytosis was also enhanced in the neurons of p35-deficient mice. Cdk5 phosphorylated the proline-rich domain of both amphiphysin I and dynamin I in vitro and in vivo. Cdk5-dependent phosphorylation of amphiphysin I inhibited the association with β-adaptin. Furthermore, the phosphorylation of dynamin I blocked its binding to amphiphysin I. The phosphorylation of each protein reduced the copolymerization into a ring formation in a cell-free system. Moreover, the phosphorylation of both proteins completely disrupted the copolymerization into a ring formation. Finally, phosphorylation of both proteins was undetectable in p35-deficient mice.

No abstract

The microtubule-associated protein Tau is a major component of the neurofibrillary tangles that serve as a neuropathological hallmark of Alzheimer's disease. Tau is a substrate for protein phosphorylation at multiple sites and occurs in tangles in a hyperphosphorylated state. However, the physiological functions of Tau phosphorylation or how it may contribute mechanistically to Alzheimer's pathophysiology are not completely understood. Here, we examined the function of human Tau phosphorylation at three sites, Ser199, Ser202, and Thr205, which together comprise the AT8 sites that mark abnormal phosphorylation in Alzheimer's disease. Overexpression of wild-type Tau or mutated forms in which these sites had been changed to either unphosphorylatable alanines or phosphomimetic aspartates inhibited mitochondrial movement in the neurite processes of PC12 cells as well as the axons of mouse brain cortical neurons. However, the greatest effects on mitochondrial translocation were induced by phosphomimetic mutations. These mutations also caused expansion of the space between microtubules in cultured cells when membrane tension was reduced by disrupting actin filaments. Thus, Tau phosphorylation at the AT8 sites may have meaningful effects on mitochondrial movement, likely by controlling microtubule spacing. Hyperphosphorylation of the AT8 sites may contribute to axonal degeneration by disrupting mitochondrial transport in Alzheimer's disease.

We tested if cannabinoid type 2 receptor (CB2) in the central nervous system plays a role in alcohol abuse/dependence in animal model and then examined an association between the CB2 gene polymorphism and alcoholism in human. Mice experiencing more alcohol preference by drinking showed reduced Cb2 gene expression, whereas mice with little preference showed no changes of it in ventral midbrain. Alcohol preference in conjunction with chronic mild stress were enhanced in mice treated with CB2 agonist JWH015 when subjected to chronic stress, whereas antagonist AM630 prevented development of alcohol preference. There is an association between the Q63R polymorphism of the CB2 gene and alcoholism in a Japanese population (P ¼ 0.007; odds ratio 1.25, 95% CI, (1.06-1.47)). CB2 under such environment is associated with the physiologic effects of alcohol and CB2 antagonists may have potential as therapies for alcoholism.

Although the roles of cyclin-dependent kinase 5 (Cdk5) in neurodevelopment and neurodegeneration have been studied extensively, regulation of Cdk5 activity has remained largely unexplored. We report here that glutamate, acting via NMDA or kainate receptors, can induce a transient Ca 2+ / calmodulin-dependent activation of Cdk5 that results in enhanced autophosphorylation and proteasome-dependent degradation of a Cdk5 activator p35, and thus ultimately down-regulation of Cdk5 activity. The relevance of this regulation to synaptic plasticity was examined in hippocampal slices using theta burst stimulation. p35 -/-mice exhibited a lower threshold for induction of long-term potentiation. Thus excitatory glutamatergic neurotransmission regulates Cdk5 activity through p35 degradation, and this pathway may contribute to plasticity.

Axonal outgrowth is a coordinated process of cytoskeletal dynamics and membrane trafficking; however, little is known about proteins responsible for regulating the membrane supply. LMTK1 (lemur kinase 1)/AATYK1 (apoptosis-associated tyrosine kinase 1) is a serine/ threonine kinase that is highly expressed in neurons. We recently reported that LMTK1 plays a role in recycling endosomal trafficking in CHO-K1 cells. Here we explore the role of LMTK1 in axonal outgrowth and its regulation by Cdk5 using mouse brain cortical neurons. LMTK1 was expressed and was phosphorylated at Ser34, the Cdk5 phosphorylation site, at the time of axonal outgrowth in culture and colocalized with Rab11A, the small GTPase that regulates recycling endosome traffic, at the perinuclear region and in the axon. Overexpression of the unphosphorylated mutant LMTK1-S34A dramatically promoted axonal outgrowth in cultured neurons. Enhanced axonal outgrowth was diminished by the inactivation of Rab11A, placing LMTK1 upstream of Rab11A. Unexpectedly, the downregulation of LMTK1 by knockdown or gene targeting also significantly enhanced axonal elongation. Rab11A-positive vesicles were transported anterogradely more quickly in the axons of LMTK1-deficient neurons than in those of wild-type neurons. The enhanced axonal outgrowth was reversed by LMTK1-WT or the LMTK1-S34D mutant, which mimics the phosphorylated state, but not by LMTK1-S34A. Thus, LMTK1 can negatively control axonal outgrowth by regulating Rab11A activity in a Cdk5-dependent manner, and Cdk5-LMTK1-Rab11 is a novel signaling pathway involved in axonal outgrowth.