Mutation of FIG4 causes neurodegeneration in the pale tremor mouse and patients with CMT4J

Membrane-bound phosphoinositides are signalling molecules that have a key role in vesicle trafficking in eukaryotic cells(1). Proteins that bind specific phosphoinositides mediate interactions between membrane-bounded compartments whose identity is partially encoded by cytoplasmic phospholipid tags. Little is known about the localization and regulation of mammalian phosphatidylinositol-3,5-bisphosphate ( PtdIns( 3,5)P-2), a phospholipid present in small quantities that regulates membrane trafficking in the endosome - lysosome axis in yeast(2). Here we describe a multi-organ disorder with neuronal degeneration in the central nervous system, peripheral neuronopathy and diluted pigmentation in the 'pale tremor' mouse. Positional cloning identified insertion of ETn2 beta ( early transposon 2 beta)(3) into intron 18 of Fig4 (A530089I17Rik), the homologue of a yeast SAC ( suppressor of actin) domain PtdIns(3,5) P-2 5-phosphatase located in the vacuolar membrane. The abnormal concentration of PtdIns( 3,5) P2 in cultured fibroblasts from pale tremor mice demonstrates the conserved biochemical function of mammalian Fig4. The cytoplasm of fibroblasts from pale tremor mice is filled with large vacuoles that are immunoreactive for LAMP-2 (lysosomal-associated membrane protein 2), consistent with dysfunction of the late endosome - lysosome axis. Neonatal neurodegeneration in sensory and autonomic ganglia is followed by loss of neurons from layers four and five of the cortex, deep cerebellar nuclei and other localized brain regions. The sciatic nerve exhibits reduced numbers of large-diameter myelinated axons, slowed nerve conduction velocity and reduced amplitude of compound muscle action potentials. We identified pathogenic mutations of human FIG4 (KIAA0274) on chromosome 6q21 in four unrelated patients with hereditary motor and sensory neuropathy. This novel form of autosomal recessive Charcot - Marie - Tooth disorder is designated CMT4J.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62835/1/nature05876.pd

Genetic interactions and functional analyses of the fission yeast gsk3 and amk2 single and double mutants defective in TORC1-dependent processes

The Target of Rapamycin (TOR) signalling network plays important roles in aging and disease. The AMP-activated protein kinase (AMPK) and the Gsk3 kinase inhibit TOR during stress. We performed genetic interaction screens using synthetic genetic arrays (SGA) with gsk3 and amk2 as query mutants, the latter encoding the regulatory subunit of AMPK. We identified 69 negative and 82 positive common genetic interactors, with functions related to cellular growth and stress. The 120 gsk3-specific negative interactors included genes functioning in translation and ribosomes. The 215 amk2-specific negative interactors included genes functioning in chromatin silencing and DNA damage repair. Both amk2- and gsk3-specific interactors were enriched in phenotype categories related to abnormal cell size and shape. We also performed SGA screen with the amk2 gsk3 double mutant as a query. Mutants sensitive to 5-fluorouracil, an anticancer drug are under-represented within the 305 positive interactors specific for the amk2 gsk3 query. The triple-mutant SGA screen showed higher number of negative interactions than the double mutant SGA screens and uncovered additional genetic network information. These results reveal common and specialized roles of AMPK and Gsk3 in mediating TOR-dependent processes, indicating that AMPK and Gsk3 act in parallel to inhibit TOR function in fission yeast

Purinergic signalling and immune cells

This review article provides a historical perspective on the role of purinergic signalling in the regulation of various subsets of immune cells from early discoveries to current understanding. It is now recognised that adenosine 5'-triphosphate (ATP) and other nucleotides are released from cells following stress or injury. They can act on virtually all subsets of immune cells through a spectrum of P2X ligand-gated ion channels and G protein-coupled P2Y receptors. Furthermore, ATP is rapidly degraded into adenosine by ectonucleotidases such as CD39 and CD73, and adenosine exerts additional regulatory effects through its own receptors. The resulting effect ranges from stimulation to tolerance depending on the amount and time courses of nucleotides released, and the balance between ATP and adenosine. This review identifies the various receptors involved in the different subsets of immune cells and their effects on the function of these cells

Apg13p and Vac8p are part of a complex of phosphoproteins that are required for cytoplasm to vacuole targeting

We have been studying protein components that function in the cytoplasm to vacuole targeting (Cvt) pathway and the overlapping process of macroautophagy. The Vac8 and Apg13 proteins are required for the import of aminopeptidase I (API) through the Cvt pathway. We have identified a protein-protein interaction between Vac8p and Apg13p by both two-hybrid and co-immunoprecipitation analysis. Subcellular fractionation of API indicates that Vac8p and Apg13p are involved in the vesicle formation step of the Cvt pathway. Kinetic analysis of the Cvt pathway and autophagy indicates that, although Vac8p is essential for Cvt transport, it is less important for autophagy, In vivo phosphorylation experiments demonstrate that both Vac8p and Apg13p are phosphorylated proteins, and Apg13p phosphorylation is regulated by changing nutrient conditions. Although Apg13p interacts with the serine/threonine kinase Apg1p, this protein is not required for phosphorylation of either Vac8p or Apg13p, Subcellular fractionation experiments indicate that Apg13p and a fraction of Apg1p are membrane-associated, Vac8p and Apg13p may be part of a larger protein complex that includes Apg1p and additional interacting proteins. Together, these components may form a protein complex that regulates the conversion between Cvt transport and autophagy in response to changing nutrient conditions