Dicty News Electronic Edition Volume 16, number 9 May 5, 2001 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at DictyBase--http://dictybase.org. ============== Abstracts ============== Involvement of a triton-insoluble floating fraction in dictyostelium cell-cell adhesion. Harris TJ, Awrey DE, Cox BJ, Ravandi A, Tsang A, Siu CH. Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6. J Biol Chem 2001 Mar 16; [epub ahead of print] We have isolated and characterized a Triton-insoluble floating fraction (TIFF) from Dictyostelium. Ten major proteins were consistently detected in TIFF and six species were identified by mass spectrometry as actin, porin, comitin, regulatory myosin light chain, a novel member of the CD36 family, and the phospholipid-anchored cell adhesion molecule gp80. TIFF was enriched with many acylated proteins. Also, the sterol: phospholipid ratio of TIFF was 10-fold higher than that of the bulk plasma membrane. Immunoelectron microscopy showed that TIFF has vesicular morphology and confirmed the association of gp80 and comitin with TIFF membranes. Several TIFF properties were similar to those of Dictyostelium contact regions, which were isolated as a cytoskeleton-associated membrane fraction. Mass spectrometry demonstrated that TIFF and contact regions shared the same major proteins. During development, gp80 colocalized with F-actin, porin and comitin at cell-cell contacts. These proteins were also recruited to gp80 caps induced by antibody crosslinking. Filipin staining revealed high sterol levels in both gp80-enriched cell-cell contacts and gp80 caps. Moreover, sterol sequestration by filipin and digitonin inhibited gp80-mediated cell-cell adhesion. This study reveals that Dictyostelium TIFF has structural properties previously attributed to vertebrate TIFF and establishes a role for Dictyostelium TIFF in cell- cell adhesion during multicellular development. ----------------------------------------------------------------------------- The MADS-box gene srfA is expressed in a complex pattern under the control of alternative promoters and is essential for different aspects of Dictyostelium development Ricardo Escalante*, Juan J. Vicente, Nicolás Moreno and Leandro Sastre Instituto de Investigaciones Biomédicas C.S.I.C/U.A.M. C/Arturo Duperier, 4, 28029 Madrid, Spain. *Author for correspondence: Fax: +34-91-585-4587. e-mail: rescalante@iib.uam.es Developmental Biology (in press) ABSTRACT srfA displays a complex temporal and cell-type specific pattern of expression in Dictyostelium and is expressed by most of its cell types at some stage of their development. This complexity is achieved by the use of alternative promoters. The promoter activity of the proximal region was found to be restricted to a subset of prestalk cells. Little or no associated expression was observed in the lower cup and basal disc during culmination. The middle promoter region was preferentially active in prestalk cells under usual conditions of filter development. Interestingly, during slug migration, the activity of this promoter in posterior prespore cells was strongly induced. The distal region displayed a dual pattern of expression. Thus, before culmination, this region drove lacZ expression in a few cells scattered along the entire structure. However, intense lacZ staining was found in the spores by the end of culmination. We have previously reported that srfA expression is essential for spore differentiation (Escalante and Sastre, 1998. Development 125, 3801-3808). Our novel finding of the expression of the gene in prestalk cells before culmination suggested that it might play additional roles in Dictyostelium development. The study of knock out strains revealed that srfA is also required for proper slug migration. Spore differentiation and slug migration defects were rescued by re-expression of srfA in the null mutant background, under the appropriate promoter control. The expression of srfA under the activity of the distal promoter region was able to rescue spore differentiation but not slug migration. Conversely, re-expression under the control of the middle promoter rescued slug morphogenesis and migration. Our results demonstrate that the correct spatial and temporal pattern of expression of srfA is essential for the different functions that this transcription factor plays in development. ----------------------------------------------------------------------------- The WASp-like Protein Scar Regulates Macropinocytosis, Phagocytosis and Endosomal Membrane Flow in Dictyostelium. David J. Seastone1, Ed Harris1, Lesly A. Temesvari2, James E. Bear 3,4, Charles L. Saxe 3, and James Cardelli 1,2,,* 1Department of Microbiology and Immunology and 2 The Feist-Weiller Cancer Center Louisiana State University Medical Center Shreveport, LA 71130 3 Department of Cell Biology Emory University School of Medicine Atlanta, GA 30322-3030 4Present address: Department of Biology Mass. Inst. Of Tech. Cambridge, MA Journal of Cell Science, in press Abstract Scar, a member of the WASp protein family, was discovered in D. discoideum during a genetic screen for second-site mutations that suppressed a developmental defect. Disruption of the scar gene led to a two-fold decrease in the levels of cellular F-actin. To investigate the role of Scar in endocytosis, phagocytosis, and endocytic membrane trafficking, processes that depend on actin polymerization, we have analyzed a Dictyostelium cell-line that is genetically null for Scar. Rates of fluid phase macropinocytosis, and phagocytosis are significantly reduced in the scar - cell-line. In addition, exocytosis of fluid phase is delayed in these cells, and movement of fluid phase from lysosomes to post-lysosomes is also delayed. Inhibition of actin polymerization with cytochalasin A resulted in similar phenotypes, suggesting that Scar-mediated polymerization of the actin cytoskeleton was important in the regulation of these processes. Supporting this conclusion, fluorescence microscopy revealed that some endo-lysosomes were ringed with F-actin in control cells but no F-actin was detected associated with endo-lysosomes in Scar null cells. Disruption of the two genes encoding the actin monomer sequestering protein profilin in wild-type cells causes defects in the rate of pinocytosis and fluid phase efflux. Consistent with a predicted physical interaction between Scar and profilin, disrupting the scar gene in the profilin null background results in greater decreases in the rate of fluid phase internalization and fluid phase release compared to either mutant alone. Taken together, these data support a model in which Scar and profilin functionally interact to regulate internalization of fluid and particles and later steps in the endosomal pathway, probably through regulation of actin cytoskeleton polymerization. ----------------------------------------------------------------------------- Rab7 Regulates Phagosome Maturation in Dictyostelium Adam Rupper1, Bryon Grove2 and James Cardelli1* Department of Microbiology and Immunology and The Feist/Weiller Cancer Center LSUHSC Shreveport, LA 71130 2Department of Anatomy and Cell Biology University of North Dakota Grand Forks, North Dakota 58202 *Corresponding Author 318-675-5756 jcarde@lsuhsc.edu Journal of Cell Science, in press Abstract A Dictyostelium Rab7 homolog has been demonstrated to regulate fluid-phase influx, efflux, retention of lysosomal hydrolases and phagocytosis. Since Rab7 function appeared to be required for efficient phagocytosis, we sought to further characterize the role of Rab7 in phagosomal maturation. Expression of GFP-Rab7 resulted in labeling of both early and late phagosomes containing yeast, but not forming phagocytic cups. In order to determine if Rab7 played a role in regulating membrane traffic between the endo/lysosomal system and maturing phagosomes, latex bead containing (LBC) phagosomes were purified from wild-type cells at various times after internalization. Glycosidases, cysteine proteinases, Rab7 and lysosomally associated membrane proteins were delivered rapidly to nascent phagosomes in control cells. LBC phagosomes isolated from cells over-expressing dominant negative (DN) Rab7 contained very low levels of LmpA (lysosomal integral membrane protein) and a-mannosidase was not detectable. Interestingly, cysteine proteinases were delivered to phagosomes as apparent pro-forms in cells over-expressing DN Rab7. Despite these defects, phagosomes in cells over-expressing DN Rab7 matured to form multi-particle spacious phagosomes, except these phagosomes remained significantly more acidic than control phagosomes. These results suggested that Rab7 regulates both an early and late step of phagosomal maturation, similar to its role in the endo/lysosomal system. ----------------------------------------------------------------------------- Phagocytosis and Macropinocytosis in Dictyostelium: Phosphoinositide-Based Processes, Biochemically Distinct James Cardelli Department of Microbiology and Immunology Feist-Weiller Cancer Center LSU Health Sciences Center Shreveport, LA 71130 Telephone: 318-675-5756 E-mail: jcarde@lsuhsc.edu Traffic, Review in Press Abstract Phagocytosis and macropinocytosis are actin-dependent clathrin-independent processes primarily performed by cells like neutrophils and macrophages that result in the internalization of particles or the formation of fluid-filled macropinosomes > 0.5 mM in diameter, respectively. Both of these processes can result in the destruction of internalized microbes, and the display of microbial antigens at the cell surface to activate T and B cells; therefore, these internalization events play critical roles in the host response to invasion by microbial pathogens. Phagocytosis consists of a number of stages, including attachment of particles to to cell surface receptors, engulfment of the particle dependent on actin polymerization and membrane exocytosis, and formation of phago-lysosomes. In contrast, the molecular steps regulating macropinocytosis are only just now being deciphered. Much remains to be learned concerning the signaling pathways that regulate these processes. Dictyostelium is a genetically and biochemically tractable professional phagocyte that has proven to be a powerful system in which to determine the nature of the molecular steps involved in regulating these internalization processes. This review primarily summarizes what is currently understood concerning the molecular mechanisms that regulate the process of phagocytosis and macropinocytosis in Dictyostelium. Recent data will also be presented that suggests that although phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) metabolism regulates both phagocytosis and macropinocytosis (a process morphologically similar to phagocytosis), the molecular components and signaling pathways regulating each pathway differ. ----------------------------------------------------------------------------- [End Dicty News, volume 16, number 9]