Dicty News Electronic Edition Volume 13, number 12 December 4, 1999 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" ============= Abstracts ============= Functional Elements Within the Dynein Microtubule-Binding Domain. Michael P. Koonce and Irina Tikhonenko Division of Molecular Medicine, Wadsworth Center Albany, NY 12201-0509 Mol. Biol. Cell. in press (February 2000) ABSTRACT. Dynein interacts with microtubules through an ATP-sensitive linkage mapped to a structurally complex region of the heavy chain following the fourth P-loop motif. Virtually nothing is known regarding how binding affinity is achieved and modulated during ATP-hydrolysis. We have performed a detailed dissection of the microtubule-contact site, using fragment expression, alanine substitution, and peptide competition. Our work identifies three clusters of amino acids important for the physical contact with microtubules; two of these fall within a region sharing sequence homology with MAP1B, the third in a region just downstream. Amino acid substitutions within any one of these regions can eliminate or weaken microtubule binding (KK3379,80, E3385, K3387, K3397, KK3410,11, W3414, RKK3418-20, F3426, R3464, S3466, K3467), suggesting that their activities are highly coordinated. A peptide that actively displaces MAP1B from microtubules perturbs dynein binding, supporting prior evidence for similar sites of interaction. We have also identified four amino acids whose substitutions affect release of the motor from the microtubule (E3413, R3444, E3460, C3469). These suggest that nucleotide-sensitive affinity may be locally controlled, at the site of contact. Our work is the first detailed description of dynein-tubulin interactions and provides a framework for understanding how affinity is achieved and modulated. ---------------------------------------------------------------------------- The Dictyostelium RasS Protein is Required for Macropinocytosis, Phagocytosis and the Control of Cell Movement Jonathan R. Chubb, Andrew Wilkins, Geraint M. Thomas and Robert H. Insall J. Cell Sci., in press. Abstract Endocytosis and cell migration both require transient localised remodelling of the cell cortex. Several lines of evidence suggest a key regulatory role in these activities for members of the Ras family of small GTPases. We have generated Dictyostelium cells lacking one member of this family, RasS, and the mutant cells are perturbed in endocytosis and cell migration. Mutant amoebae are defective in phagocytosis and fluid-phase endocytosis and are impaired in growth. Conversely, the rasS- cells show an enhanced rate of cell migration, moving three times faster than wild-type controls. The mutant cells display an aberrant morphology, are highly polarised, carry many elongated actin protrusions and show a concomitant decrease in formation of pinocytic crowns on the cell surface. These morphological aberrations are paralleled by changes in the actin cytoskeleton, with a significant proportion of the cortical F-actin relocalised to prominent pseudopodia. Rapid migration and endocytosis appear to be mutually incompatible and it is likely that RasS protein is required to maintain the normal balance between these two actin-dependent processes. ---------------------------------------------------------------------------- Dictyostelium Myosin IK is Involved in the Maintenance of Cortical Tension and Affects Motility and Phagocytosis Eva C. Schwarz, Eva M. Neuhaus, Claudia Kistler, Andreas W. Henkel, and Thierry Soldati# Department of Molecular Cell Research, Max-Planck-Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany #Corresponding author: Tel: +49-6221-486407 / Fax: +49-6221-486325 / E-mail: soldati@mzf.mpimf-heidelberg.mpg.de Journal of Cell Science, in press Summary Dictyostelium discoideum MyoK is a novel type of myosin distinguished by a remarkable architecture. MyoK is related to class I myosins but lacks a cargo-binding tail domain and carries an insertion in a surface loop suggested to modulate motor velocity. This insertion shows similarity to a secondary actin-binding site present in the tail of some class I myosins, and indeed a GST-loop construct binds actin. As a likely consequence, binding of MyoK to actin was not only ATP- but also salt-dependent. Moreover, as both binding sites reside within its motor domain and carry potential sites of regulation, MyoK might represent a new form of actin crosslinker. MyoK was distributed in the cytoplasm with a significant enrichment in dynamic regions of the cortex. Absence of MyoK resulted in a drop of cortical tension whereas overexpression led to significantly increased tension. Absence and overexpression of MyoK dramatically affected the cortical actin cytoskeleton and resulted in reduced initial rates of phagocytosis. Cells lacking MyoK showed excessive ruffling, mostly in the form of large lamellipodia, accompanied by a thicker basal actin cortex. At early stages of development, aggregation of myoK null cells was slowed due to reduced motility. Altogether, the data indicate a distinctive role for MyoK in the maintenance and dynamics of the cell cortex. ---------------------------------------------------------------------------- 5' Nucleotidase in Dictyostelium:: Protein Purification, Cloning, and Developmental Expression. Chanpen Chanchao, Can M. Eristi, Reyna Favis and Charles L. Rutherford. Biology Department, Molecular and Cellular Biology Section, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0406 Biochimica et Biophysica Acta, In Press 5' Nucleotidase (5NU) in Dictyostelium discoideum is an enzyme that shows high substrate specificity to 5'AMP. The enzyme has received considerable attention in the past because of the critical role played by cyclic AMP in cell differentiation in this organism. Degradation of cAMP by cAMP phosphodiesterase (PDE) produces 5'AMP, the substrate of 5NU. During the time course of development, the enzyme activity of 5NU increases and becomes restricted to a narrow band of cells that form the interface between the prestalk/prespore zones. We have purified a polypeptide associated with 5NU enzyme activity. Protein sequence of this peptide was obtained from Mass Spectrometry and Edman Degradation. PCR amplification of genomic DNA using degenerate oligonucleotides and a search of sequences of a cDNA project yielded DNA fragments with sequence corresponding to the peptide sequence of 5NU. In addition, a clone was found that corresponded to the classical "alkaline phosphatase" (AP) as described in several organisms. The sequences of the 5NU and AP cDNAs were not similar, indicating they are the products of separate genes and that both genes exist in Dictyostelium. Analysis of the expression of 5nu during Dictyostelium development by Northern blotting determined that the gene is developmentally regulated. Southern blot analysis showed a single form of the 5nu gene. Targeted gene disruption and knockout mutagenesis using the 5nu sequences suggested that a 5nu mutation may be lethal. ---------------------------------------------------------------------------- [End Dicty News, volume 13, number 12]