Tubulin-nucleotide interactions. Effects of removal of exchangeable guanine nucleotide on protein conformation and microtubule assembly.

The removal of tightly bound GDP from the exchangeable nucleotide-binding site of tubulin has been performed with alkaline phosphatase under conditions which essentially retain the assembly properties of the protein. When microtubule protein is treated with alkaline phosphatase, nucleotide is selectively removed from tubulin dimer rather than from MAP (microtubule-associated protein)-containing oligomeric species. Tubulin devoid of E-site (the exchangeable nucleotide-binding site of the tubulin dimer) nucleotide shows enhanced proteolytic susceptibility of the beta-subunit to thermolysin and decreased protein stability, consistent with nucleotide removal causing changes in protein tertiary structure. Pyrophosphate ion (3 mM) is able to promote formation of normal microtubules in the complete absence of GTP by incubation at 37 degrees C either with nucleotide-depleted microtubule protein or with nucleotide-depleted tubulin dimer to which MAPs have been added. The resulting microtubules contain up to 80% of tubulin lacking E-site nucleotide. In addition to its effects on nucleation, pyrophosphate competes weakly with GDP bound at the E-site. It is deduced that binding of pyrophosphate at a vacant E-site can promote microtubule assembly. The minimum structural requirement for ligands to induce tubulin assembly apparently involves charge neutralization at the E-site by bidentate ligation, which stabilizes protein domains in a favourable orientation for promoting the supramolecular protein-protein interactions involved in microtubule formation.     

High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.      

Mutations affecting embryonic cell migrations in Caenorhabditis elegans

Four recessive mutations that affect long-range embryonic migration of the two canal-associated neurons (CANs) in C. elegans were isolated and characterized with the goal of identifying genes involved in control of directed cell movement. Mutant animals were identified initially by their "withered" tails, a phenotype associated with abnormal CAN migration; the mutants were then analyzed for abnormal cell migrations by Nomarski microscopy. Based on genetic complementation tests, the mutations were assigned to four different loci, two new (mig-10 III, mig-11 III) and two previously identified (unc-39 V, vab-8 V). Mutations at all four loci affect CAN migration with high to moderate penetrance (the percentage of mutant animals that exhibit the phenotype). In addition, two other bilaterally symmetric pairs of neurons (ALM and HSN), the mesoblast M, and a pair of coelomocyte mother cells are affected by one or more of the mutations, generally with lower penetrance. With the exceptions of HSN and the right coelomocyte mother cell, which occasionally migrate beyond their normal destinations, the cells affected appear to migrate either incompletely or not at all. All the migration phenotypes show incomplete penetrance and variable expressively, although genetic tests suggest that mutations at mig-10 and vab-8 result in complete or nearly complete loss of gene function. The variability in mutant phenotypes allowed tests for interdependence of several of the affected migrations; all those analyzed appeared independent of one another. The possible nature of the mutant defects and possible roles of these four loci in cell migration are discussed.     

The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton.

The GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, motility, and cytokinesis. We recently reported on a p150 serine/threonine kinase (termed ROK alpha) binding RhoA only in its active GTP-bound state and on its cDNA; introduction of RhoA into HeLa cells resulted in translocation of the cytoplasmic kinase to plasma membranes, consistent with ROK alpha being a target for RhoA (T. Leung, E. Manser, L. Tan, and L. Lim, J. Biol. Chem. 256:29051-29054, 1995). Reanalysis of the cDNA revealed that ROK alpha contains an additional N-terminal region. We also isolated another cDNA which encoded a protein (ROK beta) with 90% identity to ROK alpha in the kinase domain. Both ROK alpha and ROK beta, which had a molecular mass of 160 kDa, contained a highly conserved cysteine/histidine-rich domain located within a putative pleckstrin homology domain. The kinases bound RhoA, RhoB, and RhoC but not Rac1 and Cdc42. The Rho-binding domain comprises about 30 amino acids. Mutations within this domain caused partial or complete loss of Rho binding. The morphological effects of ROK alpha were investigated by microinjecting HeLa cells with DNA constructs encoding various forms of ROK alpha. Full-length ROK alpha promoted formation of stress fibers and focal adhesion complexes, consistent with its being an effector of RhoA. ROK alpha truncated at the C terminus promoted this formation and also extensive condensation of actin microfilaments and nuclear disruption. The proteins exhibited protein kinase activity which was required for stress fiber formation; the kinase-dead ROK alpha K112A and N-terminally truncated mutants showed no such promotion. The latter mutant instead induced disassembly of stress fibers and focal adhesion complexes, accompanied by cell spreading. These effects were mediated by the C-terminal region containing Rho-binding, cysteine/histidine-rich, and pleckstrin homology domains. Thus, the multidomained ROK alpha appears to be involved in reorganization of the cytoskeleton, with the N and C termini acting as positive and negative regulators, respectively, of the kinase domain whose activity is crucial for formation of stress fibers and focal adhesion complexes.     

Control of nucleation in microtubule self-assembly

The inhibition of the rate and amplitude of assembly of microtubule protein at low GTP concentration is shown by measurement of microtubule length distributions to be due to the suppression of microtubule nucleation. This inhibitory effect is enhanced by GDP added before assembly, but can be overcome by a number of molecules such as pyrophosphate or ADP. The selective inhibition of nucleation by GDP in vitro, which occurs in addition to inhibition of elongation, could provide a mechanism for the control of spontaneous microtubule nucleation in vivo.     

Functionally Referential Communication in Mammals: The Past,Present and the Future

Whether animal vocalizations have the potential to communicate information regarding ongoing external events or objects has received considerable attention over the last four decades. Such ‘functionally referential signals’ (Macedonia & Evans 1993) have been shown to occur in a range of mammals and bird species and as a consequence have helped us understand the complexities that underlie animal communication, particularly how animals process and perceive their socio‐ecological worlds. Here, we review the existing evidence for functionally referential signals in mammals according to the framework put forward in the seminal Macedonia and Evans review paper. Furthermore, we elucidate the ambiguities regarding the functionally referential framework that have become obvious over the last years. Finally, we highlight new potential areas for investigation within referential signalling. We conclude the functionally referential framework is still informative when interpreting the meaning of animal vocalizations but, based on emerging research, requires further integration with other approaches investigating animal vocal complexity to broaden its applicability.     

A germline-competent embryonic stem cell line from NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice

The NOD.Cg-Prkdc ^scid Il2rg ^tm1Wjl /SzJ mouse strain, commonly known as NSG (for NOD SCID Gamma) is severely immunodeficient and thus is an excellent recipient for xenografts, and in particular for engrafting human tumor cells and human hematopoietic stem cells. In the latter case, these cells give rise to many human hematopoetic lineages in their NSG hosts, resulting in recapitulation of many of the features of a human immune system. However, the immune system of these ??humanized mice?? (huMice) is not completely functional, in part because of a lack of expression of necessary human cytokines and HLA molecules by NSG host tissues. In order to facilitate the genetic modification of this strain in order to improve the huMouse model, we have created germline competent ES cells of this strain in which such modifications can be carried out.     

Social monitoring via close calls in meerkats

Social monitoring of the actions of group members is thought to be a key development associated with group living. Humans constantly monitor the behaviour of others and respond to them in a flexible way depending on past interactions and the current social context. While other primates have also been reported to change their behaviour towards other group members flexibly based on the current state of their relationship, empirical evidence is typically linked to contextually specific events such as aggressive or reproductive interactions. In the cooperatively breeding meerkat (Suricata suricatta), we investigated whether subordinate females use frequently emitted, non-agonistic close calls to monitor the location of the dominant female and whether they subsequently adjust their response based on recent social interactions during conflict and non-conflict periods. Subjects discriminated between the close calls of the dominant female and control playbacks, responding by approaching the loudspeaker and displaying submissive behaviour only if they were currently threatened by eviction. Our results suggest that meerkats assess the risk for aggressive interactions with close associates depending on social circumstances, and respond accordingly. We argue that social monitoring based on non-agonistic cues is probably a common mechanism in group-living species that allows the adjustment of behaviour depending on variation in relationships.