Jason Burgess

Genetics of lifespan in C. elegans: molecular diversity, physiological complexity, mechanistic simplicity

The nematode Caenorhabditis elegans is used as a model system for the study of aging. Several mutant strains that have an increased lifespan have been isolated and characterized genetically and molecularly. Molecular analysis reveals that diverse types of gene products can affect worm lifespan, including proteins active in signal transduction, transcription and silencing factors, mitochondrial enzymes, and at least one protein that affects telomere length. Genetic analysis, however, suggests that these activities all converge on a few key mechanisms that impinge on lifespan, namely the production, repair and prevention of molecular damage.

AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila

Clathrin and AP-1 are required for the formation of mucin-type secretory granules in Drosophila larval salivary gland cells. Clathrin and AP-1 colocalize with secretory cargo at the trans-Golgi network (TGN) and on immature granules. Moreover, clathrin recruitment to the TGN requires AP-1. Strikingly, loss of AP-1 or clathrin profoundly blocks granule biogenesis.

Why only time will tell

The nematode Caenorhabditis elegans has become a model system for the study of the genetic basis of aging. In particular, many mutations that extend life span have been identified in this organism. When loss-of-function mutations in a gene lead to life span extension, it is a necessary conclusion that the gene normally limits life span in the wild type. The effect of a given mutation depends on a number of environmental and genetic conditions. For example, the combination of two mutations can result in additive, synergistic, subtractive, or epistatic effects on life span. Valuable insight into the processes that determine life span can be obtained from such genetic analyses, especially when interpreted with caution, and when molecular information about the interacting genes is available. Thus, genetic and molecular analyses have implicated several genes classes (daf, clk and eat) in life span determination and have indicated that aging is affected by alteration of several biological processes, namely dormancy, physiological rates, food intake, and reproduction.

PI4KIIIα is required for cortical integrity and cell polarity during Drosophila oogenesis.

ABSTRACT Phosphoinositides regulate myriad cellular processes, acting as potent signaling molecules in conserved signaling pathways and as organelle gatekeepers that recruit effector proteins to membranes. Phosphoinositide-generating enzymes have been studied extensively in yeast and cultured cells, yet their roles in animal development are not well understood. Here, we analyze Drosophila melanogaster phosphatidylinositol 4-kinase III&agr; (PI4KIII&agr;) during oogenesis. We demonstrate that PI4KIII&agr; is required for production of plasma membrane PtdIns4P and PtdIns(4,5)P2 and is crucial for actin organization, membrane trafficking and cell polarity. Female germ cells mutant for PI4KIII&agr; exhibit defects in cortical integrity associated with failure to recruit the cytoskeletal-membrane crosslinker Moesin and the exocyst subunit Sec5. These effects reflect a unique requirement for PI4KIII&agr;, as egg chambers from flies mutant for either of the other Drosophila PI4Ks, fwd or PI4KII, show Golgi but not plasma membrane phenotypes. Thus, PI4KIII&agr; is a vital regulator of a functionally distinct pool of PtdIns4P that is essential for PtdIns(4,5)P2-dependent processes in Drosophila development.

The Garz Sec7 domain guanine nucleotide exchange factor for Arf regulates salivary gland development in Drosophila.

Surface delivery of proteins involved in cell-cell and cell-matrix interactions in cultured mammalian cells requires the GBF1 guanine nucleotide exchange factor. However, the role of GBF1 in delivery of adhesion proteins during organogenesis in intact animals has not been characterized. Here, we report the function of the fly GBF1 homolog, Gartenzwerg (Garz) in the development of the salivary gland in Drosophila melanogaster. We used the GAL4/UAS system to selectively deplete Garz from salivary gland cells. We show that depletion of Garz disrupts the secretory pathway as evidenced by the collapse of Golgi-localized Lava lamp (Lva) and the TGN-localized gamma subunit of the clathrin-adaptor protein complex (AP-1). Additionally, Garz depletion inhibits trafficking of cell-cell adhesion proteins cadherin (DE-cad) and Flamingo to the cell surface. Disregulation of trafficking correlates with mistargeting of the tumor suppressor protein Discs large involved in epithelial polarity determination. Garz-depleted salivary cells are smaller and lack well-defined plasma membrane domains. Garz depletion also inhibits normal elongation and positioning of epithelial cells, resulting in a disorganized salivary gland that lacks a well defined luminal duct. Our findings suggest that Garz is essential for establishment of epithelial structures and demonstrate an absolute requirement for Garz during Drosophila development.

Type II phosphatidylinositol 4-kinase regulates nerve terminal growth and synaptic vesicle recycling

Abstract Type II phosphatidylinositol 4-kinase (PI4KII) is thought to be associated with synaptic vesicles (SVs) and to be responsible for the majority of PI4K activity in the nervous system. However, the function of PI4KII at the synapse is unknown. We characterized the synaptic phenotypes of a Drosophila melanogaster PI4KII null mutant. We found increased nerve terminal growth in PI4KII null mutants indicating that PI4KII restrains nerve terminal growth. Evoked neurotransmitter release elicited in response to low frequency stimulation and spontaneous neurotransmitter release were not altered in PI4KII null mutants. However, PI4KII null mutants displayed reduced FM1-43 uptake in response to stimulation by high K+ saline, indicating impaired SV endocytosis. PI4KII null mutants did not display any defects in FM1-43 unloading, consistent with normal SV exocytosis. Thus, PI4KII is required for SV endocytosis but dispensable for SV exocytosis. Overall, our data show that PI4KII regulates both nerve terminal growth and SV recycling.

The phosphoinositide phosphatase Sac1 regulates cell shape and microtubule stability in the developing Drosophila eye

ABSTRACT Epithelial patterning in the developing Drosophila melanogaster eye requires the Neph1 homolog Roughest (Rst), an immunoglobulin family cell surface adhesion molecule expressed in interommatidial cells (IOCs). Here, using a novel temperature-sensitive (ts) allele, we show that the phosphoinositide phosphatase Sac1 is also required for IOC patterning. Sac1ts mutants have rough eyes and retinal patterning defects that resemble rst mutants. Sac1ts retinas exhibit elevated levels of phosphatidylinositol 4-phosphate (PI4P), consistent with the role of Sac1 as a PI4P phosphatase. Indeed, genetic rescue and interaction experiments reveal that restriction of PI4P levels by Sac1 is crucial for normal eye development. Rst is delivered to the cell surface in Sac1ts mutants. However, Sac1ts mutant IOCs exhibit severe defects in microtubule organization, associated with accumulation of Rst and the exocyst subunit Sec8 in enlarged intracellular vesicles upon cold fixation ex vivo. Together, our data reveal a novel requirement for Sac1 in promoting microtubule stability and suggest that Rst trafficking occurs in a microtubule- and exocyst-dependent manner. Summary: Analysis of temperature-sensitive mutants identifies a novel role for the lipid phosphatase Sac1 in regulating epithelial patterning and microtubule organization during early stages of Drosophila eye development.