Radial compression of microtubules and the mechanism of action of taxol and associated proteins

Microtubules (MTs) are hollow cylindrical polymers composed of alphabeta-tubulin heterodimers that align head-to-tail in the MT wall, forming linear protofilaments that interact laterally. We introduce a probe of the interprotofilament interactions within MTs and show that this technique gives insight into the mechanisms by which MT-associated proteins (MAPs) and taxol stabilize MTs. In addition, we present further measurements of the mechanical properties of MT walls, MT-MT interactions, and the entry of polymers into the MT lumen. These results are obtained from a synchrotron small angle x-ray diffraction (SAXRD) study of MTs under osmotic stress. Above a critical osmotic pressure, P(cr), we observe rectangular bundles of MTs whose cross sections have buckled to a noncircular shape; further increases in pressure continue to distort MTs elastically. The P(cr) of approximately 600 Pa provides, for the first time, a measure of the bending modulus of the interprotofilament bond within an MT. The presence of neuronal MAPs greatly increases P(cr), whereas surprisingly, the cancer chemotherapeutic drug taxol, which suppresses MT dynamics and inhibits MT depolymerization, does not affect the interprotofilament interactions. This SAXRD-osmotic stress technique, which has enabled measurements of the mechanical properties of MTs, should find broad application for studying interactions between MTs and of MTs with MAPs and MT-associated drugs.     

A multicopper ferroxidase involved in iron binding to transferrins in Dunaliella salina plasma membranes

The halotolerant alga Dunaliella salina is unique among plants in that it utilizes a transferrin (TTf) to mediate iron acquisition (Fisher, M., Zamir, A., and Pick, U. (1998) J. Biol. Chem. 273, 17553-17558). Two new proteins that are induced by iron deprivation were identified in plasma membranes of D. salina as follows: a multicopper ferroxidase termed D-Fox and an internally duplicated glycoprotein (p130B). D-Fox and p130B are accessible to glycolytic, proteolytic, and biotin surface tagging treatments, suggesting that they are surface-exposed glycoproteins. Induction of D-Fox was also manifested by ferroxidase activity in plasma membrane preparations. These results are puzzling because ferroxidases in yeast and in Chlamydomonas reinhardtii function in redox-mediated iron uptake, a mechanism that is not known to operate in D. salina. Two lines of evidence suggest that D-Fox and p130B interact with D. salina triplicated transferrin (TTf). First, chemical cross-linking combined with mass spectroscopy analysis showed that D-Fox and p130B associate with TTf and with another plasma membrane transferrin. Second, detergent-solubilized D-Fox and p130B comigrated on blue native gels with plasma membrane transferrins. 59Fe autoradiography indicated that this complex binds Fe3+ ions. Also, the induction of D-Fox and p130B is kinetically correlated with enhanced iron binding and uptake activities. These results suggest that D-Fox and p130B associate with plasma membrane transferrins forming a complex that enhances iron binding and iron uptake. We propose that the function of D-Fox in D. salina has been modified during evolution from redox-mediated to transferrin-mediated iron uptake, following a gene transfer event of transferrins from an ancestral animal cell.     

Hippocampal "time cells" bridge the gap in memory for discontiguous events

The hippocampus is critical to remembering the flow of events in distinct experiences and, in doing so, bridges temporal gaps between discontiguous events. Here, we report a robust hippocampal representation of sequence memories, highlighted by "time cells" that encode successive moments during an empty temporal gap between the key events, while also encoding location and ongoing behavior. Furthermore, just as most place cells "remap" when a salient spatial cue is altered, most time cells form qualitatively different representations ("retime") when the main temporal parameter is altered. Hippocampal neurons also differentially encode the key events and disambiguate different event sequences to compose unique, temporally organized representations of specific experiences. These findings suggest that hippocampal neural ensembles segment temporally organized memories much the same as they represent locations of important events in spatially defined environments.     

Semihemoglobins, high oxygen affinity dimeric forms of human hemoglobin respond efficiently to allosteric effectors without forming tetramers

Significant reduction in oxygen affinity resulting from interactions between heterotropic allosteric effectors and hemoglobin in not only the unligated derivative but also the fully ligated form has been reported (Tsuneshige, A., Park, S. I., and Yonetani, T. (2002) Biophys. Chem. 98, 49-63; Yonetani, T., Park, S. I., Tsuneshige, A., Imai, K., and Kanaori, K. (2002) J. Biol. Chem. 277, 34508-34520). To further investigate this effect in more detail, alpha- and beta-semihemoglobins, namely, alpha(heme)beta(apo) and alpha(apo)beta(heme), respectively, were prepared and characterized with respect to the impact of allosteric effectors on both conformation and ligand binding properties. Semihemoglobins are dimers characterized by a high affinity for oxygen and lack of cooperativity. We found that, compared with stripped conditions, semihemoglobins responded to effectors (inositol hexaphosphate and L35) by decreasing the affinity for oxygen by 60- and 130-fold for alpha- and beta-semihemoglobins, respectively. 1H NMR and sedimentation velocity experiments carried out with their ligated and unligated forms in the absence and presence of effectors revealed that semihemoglobins always remain as single-heme-carrying dimers. Recombination kinetics of their photolyzed CO derivatives showed that effectors did indeed interact with their ligated forms. Measurements of the Fe-His stretching mode show that the semihemoglobins undergo a large ligand binding-induced conformational shift and that both ligand-free and ligand derivatives respond to the presence of effectors. Contradictions to the Monod-Wyman-Changeaux/Perutz allosteric model arise since 1) the modulation of ligand affinity is not achieved in semihemoglobins by the formation of a low affinity T conformation (quaternary effect) but by direct interaction with effectors, 2) effectors do interact significantly with ligated forms of high affinity semihemoglobins, and 3) modulation of the ligand affinity and the cooperativity are not necessarily linked but instead can be separated into two distinct phenomena that can be isolated.     

RAFTK/Pyk2 activation is mediated by trans-acting autophosphorylation in a Src-independent manner

The related adhesion focal tyrosine kinase (RAFTK), also known as Pyk2, undergoes autophosphorylation upon its stimulation. This leads to cascades of intracellular signaling that result in the regulation of various cellular activities. However, the molecular mechanism of RAFTK autophosphorylation is not yet known. Using various RAFTK constructs fused with two different tags, we found that the autophosphorylation of RAFTK was mediated by a trans-acting mechanism, not a cis-acting mechanism. In addition, overexpression of kinase-mutated RAFTK inhibited wild type RAFTK autophosphorylation in a dose-dependent manner by a trans-acting interaction. Trans-acting autophosphorylation was also observed between endogenous and exogenous RAFTK upon potassium depolarization of neuroendocrine PC12 cells. Using immunoprecipitation and affinity chromatography, we detected RAFTK self-association that was not affected by deletion of a single region or domain of RAFTK. Furthermore, RAFTK autophosphorylation occurred only at site Tyr402 in a Src kinase activity-independent manner. However, Src significantly enhanced RAFTK-mediated paxillin phosphorylation, suggesting a key role for Src in RAFTK activation and phosphorylation of downstream substrates. Our results indicate that the activation of RAFTK occurs in several steps. First, upon stimulus, RAFTK trans-autophosphorylates Tyr402. Second, phosphorylated Tyr402 recruits and activates Src kinase that in turn phosphorylates RAFTK and enhances its kinase activity. Lastly, the enhanced RAFTK activity induces the activation of downstream signaling molecules. Taken together, these studies provide insights into the molecular mechanism of RAFTK autophosphorylation and the specific role of Src in the regulation of RAFTK activation.