University of Texas at Austin
School of Biological Sciences The Section of Molecular Cell and Developmental Biology The Institute for Cellular and Molecular Biology School of Biological Sciences / The Section of Molecular Cell and Developmental Biology / The Institute for Cellular and Molecular Biology
Research

Current Research Projects in the Lab:
Mechanisms of membrane transport required for cell formation.

Through our studies of the golgin protein Lava Lamp (LVA) we have established that a significant amount of the plasma membrane growth required for cell formation in Drosophila embryos requires de novo Golgi-dependent membrane secretion (Papoulas et al., 2005; Sisson et al., 2000). Golgin proteins were originally identified as human autoantigens implicated in a variety of human autoimmune diseases (Chan and Fritzler, 1998) and are thought to function on the surface of Golgi bodies as scaffolds for Golgi structural integrity and tethers for specific membrane transport vesicles or the cytoskeleton (Short et al., 2005). We have shown that LVA functions as an adaptor for the microtubule (MT) motility factors cytoplasmic Dynein, Dynactin, and CLIP-190, the Drosophila ortholog of CLIP-170 (Figure 2)(Papoulas et al., 2005). Together these proteins target Golgi bodies towards the minus-ends of MTs and the embryo’s surface to facilitate secretion of new plasma membrane required for cleavage furrow formation (view movie pop video window)(Papoulas et al., 2005). One of our current research priorities is to test whether LVA functions as a molecular switch to turn on and off Dynactin-dependent, Dynein-driven movement of Golgi bodies in live embryos.
  Figure 2.
 
Figure 2. (click for details)
Figure 2. A model showing Lava Lamp functioning as an adaptor for minus-end directed microtubule motility factors. Black arrows symbolize observed interactions between the numbered segments of Lava Lamp and the indicated components. Gray arrows indicate proposed interactions. A single microtubule (MT) is depicted with labeled plus (+) end. From Papoulas, et al., 2005.
 

Figure 3. We have also begun to investigate the role of LVA during later stages in development. These studies have demonstrated that LVA is required in somatic cells for viability and female fertility. We know that diminished LVA activity in somatic cells of adult females blocks oogenesis, and we are now in the process of establishing the cellular basis of this effect (Figure 3).

Figure 3. (click for details)
Figure 3. Lava Lamp is ubiquitously expressed in ovaries. This laser-scanning confocal micrograph of a fixed wild type egg chamber reveals the position of nuclei (circular green structures) and the subcellular localization of microfilaments (blue) and Lava Lamp (red) in germ line nurse cells (nc) and oocyte (oo), and somatic follicle cells (fc). Scale bar represents 50 μm.
 

Figure 4. A screen of an existing collection of temperature sensitive (ts) mutants for defects in cleavage furrow formation using time-lapse differential interference contrast (DIC) microscopy of live mutant embryos has identified at least one gene required for membrane secretion. A total of five mutants were found that display robust defects within minutes of shifting live embryos to the restrictive temperature. Four of these mutations exist in genes that have not been previously implicated in cleavage furrow formation. Our analysis of one of these four mutants, which we have named inferno, suggests it disrupts the early secretory pathway (Figure 4). Our future characterization of inferno and the other four mutants should provide a more complete understanding of the molecular mechanisms controlling cleavage furrow formation. Drs. Helen Francis-Lang and William Sullivan kindly provided the mutant collection for this project.

Figure 4. (click for details)
Figure 4. De novo secretion of Neurotactin protein is disrupted in inferno mutant embryos at the restrictive temperature. Lasar-scanning confocal micrographs of WT (A and B) and inferno (C and D) cellularizing embryos fixed at 32°C. Neurotactin fails to accumulate in furrows (compare A and C) and a general disruption in furrow organization (compare B and D) is observed in inferno mutants by immunofluorescence and fluorescent-ConA detection. ConA is binding glycoproteins and glycolipids on the plasma membrane. Arrowheads indicate the furrow front position and arrows indicate ectopic ConA detected at the furrow front. Scale bar represents 10 μm.
 

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