Stathmin strongly increases the minus end catastrophe frequency and induces rapid treadmilling of bovine brain microtubules at steady state in vitro

Stathmin is a ubiquitous microtubule destabilizing protein that is believed to play an important role linking cell signaling to the regulation of microtubule dynamics. Here we show that stathmin strongly destabilizes microtubule minus ends in vitro at steady state, conditions in which the soluble tubulin and microtubule levels remain constant. Stathmin increased the minus end catastrophe frequency approximately 13-fold at a stathmin:tubulin molar ratio of 1:5. Stathmin steady-state catastrophe-promoting activity was considerably stronger at the minus ends than at the plus ends. Consistent with its ability to destabilize minus ends, stathmin strongly increased the treadmilling rate of bovine brain microtubules. By immunofluorescence microscopy, we also found that stathmin binds to purified microtubules along their lengths in vitro. Co-sedimentation of purified microtubules polymerized in the presence of a 1:5 initial molar ratio of stathmin to tubulin yielded a binding stoichiometry of 1 mol of stathmin per approximately 14.7 mol of tubulin in the microtubules. The results firmly establish that stathmin can increase the steady-state catastrophe frequency by a direct action on microtubules, and furthermore, they indicate that an important regulatory action of stathmin in cells may be to destabilize microtubule minus ends.     

The intracellular chloride ion channel protein CLIC1 undergoes a redox-controlled structural transition

Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24-Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity.     

Homomeric ring assemblies of eukaryotic Sm proteins have affinity for both RNA and DNA. Crystal structure of an oligomeric complex of yeast SmF

Sm and Sm-like proteins are key components of small ribonucleoproteins involved in many RNA and DNA processing pathways. In eukaryotes, these complexes contain seven unique Sm or Sm-like (Lsm) proteins assembled as hetero-heptameric rings, whereas in Archaea and bacteria six or seven-membered rings are made from only a single polypeptide chain. Here we show that single Sm and Lsm proteins from yeast also have the capacity to assemble into homo-oligomeric rings. Formation of homo-oligomers by the spliceosomal small nuclear ribonucleoprotein components SmE and SmF preclude hetero-interactions vital to formation of functional small nuclear RNP complexes in vivo. To better understand these unusual complexes, we have determined the crystal structure of the homomeric assembly of the spliceosomal protein SmF. Like its archaeal/bacterial homologs, the SmF complex forms a homomeric ring but in an entirely novel arrangement whereby two heptameric rings form a co-axially stacked dimer via interactions mediated by the variable loops of the individual SmF protein chains. Furthermore, we demonstrate that the homomeric assemblies of yeast Sm and Lsm proteins are capable of binding not only to oligo(U) RNA but, in the case of SmF, also to oligo(dT) single-stranded DNA.     

The phosphorylation of stathmin by MAP kinase

Stathmin, a ubiquitous cytosolic phosphoprotei which may play a role in integrating the effects of diverse signals regulating proliferation, differentiation and other cell functions, was found to be phosphorylated rapidly and stoichiometrically by mitogen-activated protein (MAP) kinasein vitro. Ser-25 was identified as the major site and Ser-38 as a minor site of phosphorylation, while the p42 and p44 isoforms of MAP kinase were the only significant stathmin kinases detected in PC12 cells after stimulation by nerve growth factor (NGF). The results suggest that MAP kinases are the enzymes responsible for increasing the level of phosphorylation of Ser-25, which has been observed previously in PC12 cells following stimulation by NGF.Submitted February 1993.     

Coherent phenomena in photosynthetic light harvesting: part one—theory and spectroscopy

The role of non-trivial quantum mechanical effects in biology has been the subject of intense scrutiny over the past decade. Much of the focus on potential “quantum biology” has been on energy transfer processes in photosynthetic light harvesting systems. Ultrafast laser spectroscopy of several light harvesting proteins has uncovered coherent oscillations dubbed “quantum beats” that persist for hundreds of femtoseconds and are putative signatures for quantum transport phenomena. This review describes the language and basic quantum mechanical phenomena that underpin quantum transport in open systems such as light harvesting and photosynthetic proteins, including the photosystem reaction centre. Coherent effects are discussed in detail, separating various meanings of the term, from delocalized excitations, or excitons, to entangled states and coherent transport. In particular, we focus on the time, energy and length scales of energy transport processes, as these are critical in understanding whether or not coherent processes are important. The role played by the protein in maintaining chromophore systems is analysed. Finally, the spectroscopic techniques that are used to probe energy transfer dynamics and that have uncovered the quantum beats are described with reference to coherent phenomena in light harvesting.     

Molecular Interactions of the Min Protein System Reproduce Spatiotemporal Patterning in Growing and Dividing Escherichia coli Cells

Oscillations of the Min protein system are involved in the correct midcell placement of the divisome during Escherichia coli cell division. Based on molecular interactions of the Min system, we formulated a mathematical model that reproduces Min patterning during cell growth and division. Specifically, the increase in the residence time of MinD attached to the membrane as its own concentration increases, is accounted for by dimerisation of membrane-bound MinD and its interaction with MinE. Simulation of this system generates unparalleled correlation between the waveshape of experimental and theoretical MinD distributions, suggesting that the dominant interactions of the physical system have been successfully incorporated into the model. For cells where MinD is fully-labelled with GFP, the model reproduces the stationary localization of MinD-GFP for short cells, followed by oscillations from pole to pole in larger cells, and the transition to the symmetric distribution during cell filamentation. Cells containing a secondary, GFP-labelled MinD display a contrasting pattern. The model is able to account for these differences, including temporary midcell localization just prior to division, by increasing the rate constant controlling MinD ATPase and heterotetramer dissociation. For both experimental conditions, the model can explain how cell division results in an equal distribution of MinD and MinE in the two daughter cells, and accounts for the temperature dependence of the period of Min oscillations. Thus, we show that while other interactions may be present, they are not needed to reproduce the main characteristics of the Min system in vivo.     

Rapid assembly and collective behavior of microtubule bundles in the presence of polyamines

Microtubules (MTs) are cylindrical cytoskeleton polymers composed of α-β tubulin heterodimers whose dynamic properties are essential to fulfill their numerous cellular functions. In response to spatial confinement, dynamic MTs, even in the absence of protein partners, were shown to self-organize into higher order structures (spindle or striped structures) which lead to interesting dynamical properties (MT oscillations). In this study, we considered the assembly and sensitivity of dynamic MTs when in bundles. To perform this study, spermine, a natural tetravalent polyamine present at high concentrations in all eukaryote cells, was used to trigger MT bundling while preserving MT dynamics. Interestingly, we first show that, near physiological ionic strengths, spermine promotes the bundling of MTs whereas it does not lead to aggregation of free tubulin, which would have been detrimental to MT polymerization. Experimental and theoretical results also indicate that, to obtain a high rate of bundle assembly, bundling should take place at the beginning of assembly when rapid rotational movements of short and newly nucleated MTs are still possible. On the other hand, the bundling process is significantly slowed down for long MTs. Finally, we found that short MT bundles exhibit a higher sensitivity to cold exposure than do isolated MTs. To account for this phenomenon, we suggest that a collective behavior takes place within MT bundles because an MT entering into a phase of shortening could increase the probability of the other MTs in the same bundle to enter into shortening phase due to their close proximity. We then elaborate on some putative applications of our findings to in vivo conditions including neurons.     

Systematics, biostratigraphy and evolutionary pattern of the Oligo-Miocene marine mammals from the Maltese Islands

An overview of the upper Oligocene-upper Miocene marine sediments outcropping in the Maltese Islands provides a detailed stratigraphical setting of several marine mammal assemblages. The studied fossil material collected within the entire sequence, is now kept in the National Museum of Natural History of Mdina (Malta). Nannoplankton analysis of some selected sections, where mammal remains have been discovered, is also undertaken. The fossil marine mammals, consisting mostly of isolated ear bones and teeth, are referred to cetaceans (both mysticetes and odontocetes), sirenians, and pinnipeds. The cetacean record evidences an evolutionary pattern that agrees with the Oligo-Miocene general trend, characterized by the progressive rarefaction and disappearance of archaic families (squalodontids, waipatiids, and, maybe, mammalodontids), and by the appearance and diversification of the extant families represented within younger strata (kogiids, pontoporiids and ziphiids). Pontoporiids, waipatiids, and tentatively mammalodontids are here reported for the first time in the Mediterranean, while the kogiid record represents the only sure Miocene evidence of this family in the Mediterranean. The geographical distribution of the mammalodontids and the waipatiids, based on the Maltese and extra-Mediterranean records, supports an open communication between the Proto-Mediterranean and the Indo-Pacific during the late Oligocene. Sirenians are represented by several dugongid pachyosteosclerotic rib fragments, collected from upper Oligocene through upper Miocene sediments. Pinnipeds are represented by a femur fragment from the Serravallian, referred to an indeterminate monachine, a phocid subfamily already reported from the Mio-Pliocene of the Mediterranean.     

A putative house-cleaning enzyme encoded within an integron array: 1.8 A crystal structure defines a new MazG subtype

Mobile gene cassettes collectively contain a highly diverse pool of novel genes that encode many novel adaptive functions. In the non-clinical context, the function of almost all of the encoded proteins remains unknown despite the enormous size of this mobile gene pool. We have been characterizing cassette arrays by taking advantage of the fact that they cluster at discrete sites in chromosomes; even large arrays are thus recoverable in a relatively small number of clones in genomic libraries. In one assembled array of 116 cassettes from the marine bacterium Vibrio sp. DAT722, a putative MazG protein is encoded within the 21st cassette. Because MazG proteins are implicated in a number of cellular processes, including house-cleaning and stress survival, the presence of such a protein in a mobile cassette was noteworthy. Here we solve the crystal structure of this α-helical protein, and define both open and closed states of a new variant of the MazG family. Functional assays confirm that the protein is a dNTP pyrophosphohydrolase, with marked preferences for dCTP and dATP. We hypothesize that i MazG acts as a house-cleaning enzyme, preventing the incorporation of damaging non-canonical nucleotides into host-cell DNA.