Kif18B interacts with EB1 and controls astral microtubule length during mitosis

Regulation of microtubule (MT) dynamics is essential for proper spindle assembly and organization. Kinesin-8 family members are plus-end-directed motors that modulate plus-end MT dynamics by acting as MT depolymerases or as MT plus-end capping proteins. In this paper, we show that the human kinesin-8 Kif18B functions during mitosis to control astral MT organization. Kif18B is a MT plus-tip-tracking protein that localizes to the nucleus in interphase and is enriched at astral MT plus ends during early mitosis. Knockdown of Kif18B caused spindle defects, resulting in an increased number and length of MTs. A yeast two-hybrid screen identified an interaction of the C-terminal domain of Kif18B with the plus-end MT-binding protein EB1. EB1 knockdown disrupted Kif18B targeting to MT plus ends, indicating that EB1/Kif18B interaction is physiologically important. This interaction is direct, as the far C-terminal end of Kif18B is sufficient for binding to EB1 in vitro. Overexpression of this domain is sufficient for plus-end MT targeting in cells; however, targeting is enhanced by the motor domain, which cooperates with the tail to achieve proper Kif18B localization at MT plus ends. Our results suggest that Kif18B is a new MT dynamics regulatory protein that interacts with EB1 to control astral MT length.     

ZnT5 variant B is a bidirectional zinc transporter and mediates zinc uptake in human intestinal Caco-2 cells

Zinc is an essential micronutrient, so it is important to elucidate the molecular mechanisms of zinc homeostasis, including the functional properties of zinc transporters. Mammalian zinc transporters are classified in two major families: the SLC30 (ZnT) family and the SLC39 family. The prevailing view is that SLC30 family transporters function to reduce cytosolic zinc concentration, either through efflux across the plasma membrane or through sequestration in intracellular compartments, and that SLC39 family transporters function in the opposite direction to increase cytosolic zinc concentration. We demonstrated that human ZnT5 variant B (ZnT5B (hZTL1)), an isoform expressed at the plasma membrane, operates in both the uptake and the efflux directions when expressed in Xenopus laevis oocytes. We measured increased activity of the zinc-responsive metallothionein 2a (MT2a) promoter when ZnT5b was co-expressed with an MT2a promoter-reporter plasmid construct in human intestinal Caco-2 cells, indicating increased total intracellular zinc concentration. Increased cytoplasmic zinc concentration mediated by ZnT5B, in the absence of effects on intracellular zinc sequestration by the Golgi apparatus or endoplasmic reticulum, was also confirmed by a dramatically enhanced signal from the zinc fluorophore Rhodzin-3 throughout the cytoplasm of Caco-2 cells overexpressing ZnT5B at the plasma membrane when compared with control cells. Our findings demonstrate clearly that, in addition to mediating zinc efflux, ZnT5B at the plasma membrane can function to increase cytoplasmic zinc concentration, thus indicating a need to reevaluate the current paradigm that SLC30 family zinc transporters operate exclusively to decrease cytosolic zinc concentration.     

Regulation of microtubule dynamics by TOG-domain proteins XMAP215/Dis1 and CLASP

The molecular mechanisms by which microtubule-associated proteins (MAPs) regulate the dynamic properties of microtubules (MTs) are still poorly understood. We review recent advances in our understanding of two conserved families of MAPs, the XMAP215/Dis1 and CLASP family of proteins. In vivo and in vitro studies show that XMAP215 proteins act as microtubule polymerases at MT plus ends to accelerate MT assembly, and CLASP proteins promote MT rescue and suppress MT catastrophe events. These are structurally related proteins that use conserved TOG domains to recruit tubulin dimers to MTs. We discuss models for how these proteins might use these individual tubulin dimers to regulate dynamic behavior of MT plus ends.     

When MT1-MMP meets ADAMs

MT1-MMP is a membrane-tethered enzyme capable of remodeling extracellular matrix. MT1-MMP-deficient mice exhibit systematic defects during development, especially in craniofacial development characterized by retarded calvarial bone formation. Recently, we identified MT1-MMP as a critical positive modulator of FGF signaling during intramembranous ossification. MT1-MMP cleaves ADAM9 to protect FGFR2 from ectodomain shedding. Depletion of ADAM9 in MT1-MMP-deficient mice significantly rescued the calvarial defects via restoring FGF signaling. Interestingly, this regulatory mechanism seems to be highly tissue-specific, as defective FGF2-induced corneal angiogenesis in Mmp14^?/? mice could not be rescued by removal of ADAM9. In addition, MT1-MMP also cleaves another ADAM family member, ADAM15. Our current findings not only present a novel regulatory mechanism for FGF signaling but also reveal a functional crosstalk between MMP and ADAM families. Better understanding of the interplay between ADAMs and MT1-MMP and its consequences for signaling pathways will provide new insights into therapeutic approaches for the management of developmental disorders and various diseases, such as cancer.     

The plant B3 superfamily

The plant-specific B3 superfamily encompasses well-characterized families, such as the auxin response factor (ARF) family and the LAV family, as well as less well understood families, such as RAV and REM. In Arabidopsis, there are 118 B3 genes, and in rice there are 91 B3 genes. The B3 domain is present in genes from gymnosperms, mosses and green algae, indicating that the B3 domain evolved on the plant lineage before multicellularity. The aim of this review is to phylogenetically characterize the members of the B3 family in Arabidopsis and rice and to review the function of the B3 genes that have been studied to date.     

Catalytic activities of membrane-type 6 matrix metalloproteinase (MMP25)

This study describes the biochemical characterisation of the catalytic domain of membrane-type 6 matrix metalloproteinase (MT6-MMP, MMP25, leukolysin). Its activity towards synthetic peptide substrates, components of the extracellular matrix and inhibitors of MMPs was studied and compared with MT1-MMP, MT4-MMP and stromelysin-1. We have found that MT6-MMP is closer in function to stromelysin-1 than MT1 and MT4-MMP in terms of substrate and inhibitor specificity, being able to cleave type-IV collagen, gelatin, fibronectin and fibrin. However, it differs from stromelysin-1 and MT1-MMP in its inability to cleave laminin-I, and unlike stromelysin-1 cannot activate progelatinase B. Our findings suggest that MT6-MMP could play a role in cellular migration and invasion of the extracellular matrix and basement membranes and its activity may be tightly regulated by all members of the TIMP family.     

Sex ratios in offspring of sex-reversed sea bass and the relationship between growth and phenotypic sex differentiation

Groups of sexually undifferentiated sea bass Dicentrarchus labrax were fed with the androgen 17α-methyltestosterone (MT) during sex differentiation. MT treatment increased males from 79±3% in the controls (the usual 3:1 male:female sex ratio of cultured sea bass) to 100±0%, implying that in the treated groups one out of each five resulting males was a masculinized female (neomale). Thirteen males from the MT treated groups were taken as the parental generation and their sperm used to individually fertilize a pool of eggs from unrelated females. The probability of having at least one neomale was 95% and most probably two or three of the males used were neomales. The offspring from each family were reared separately under the same environmental conditions. Samples were taken at 11 and 15 months of age, during and after sex differentiation, respectively. Results showed that females predominated among the larger fish whereas males and undifferentiated fish predominated among the smaller ones. Intersexes exhibited an intermediate size. All fish with a body length smaller than 12 cm were undifferentiated. These results suggest that sex differentiation is more dependent on length than on age. At 15 months, sex ratios were male-biased in all families, except one (females ranged from 5 to 50%) and only two families had sex ratios not significantly different from 1:1, suggesting that the mechanism of sex determination in the sea bass is not of a XX/XY or ZW/ZZ type since no family exhibited a female-biased progeny, as would be expected from both types. Results support the hypothesis that factors other than genetic, i.e., environmental, may act epigenetically on the sex determination mechanisms of sea bass, as has been demonstrated in other fishes.     

Antibody MT3 is reactive with a novel exon B-associated 190-kDa sialic acid-dependent epitope of the leukocyte common antigen complex.

MT3 is a new antibody reactive with a restricted isoform of the leukocyte common Ag (CD45) family. The Ag is mainly expressed on T lymphocytes and thymocytes. It is differentially expressed on B cell subpopulations, with no staining of the majority of small follicular mantle zone B cells but positive staining of the majority of marginal zone B cells of spleen. Like most CD45 antibodies, reactivity can be demonstrated in fresh frozen as well as in formalin-fixed, paraffin-embedded tissues. This reactivity clearly differs from all other published anti-CD45 antibodies. In immunoprecipitation and Western blot procedures, the antibody reacts with a major band with a molecular mass of 190 kDa and weak bands with molecular masses of 205 and 220 kDa. Compared to antibody PD7 that reacts with exon B-encoded sequences, the reactivity with the 205 and 220 bands is much weaker. This is reflected in MT3 reactivity with leukocyte common Ag transfectants that include exon B-encoded sequences, such as AB and B, but not with those that also include C-encoded sequences, such as ABC or BC. It can be concluded that MT3 recognizes additional heterogeneity in the leukocyte common Ag complex, that is based on the differential expression of sialic acid-dependent determinants associated with exon B-encoded sequences.     

Metallothionein and a peptide modeled after metallothionein, EmtinB, induce neuronal differentiation and survival through binding to receptors of the low-density lipoprotein receptor family

Accumulating evidence suggests that metallothionein (MT)-I and -II promote neuronal survival and regeneration in vivo . The present study investigated the molecular mechanisms underlying the differentiation and survival-promoting effects of MT and a peptide modeled after MT, EmtinB. Both MT and EmtinB directly stimulated neurite outgrowth and promoted survival in vitro using primary cultures of cerebellar granule neurons. In addition, expression and surface localization of megalin, a known MT receptor, and the related lipoprotein receptor-related protein-1 (LRP) are demonstrated in cerebellar granule neurons. By means of surface plasmon resonance MT and EmtinB were found to bind to both megalin and LRP. The bindings were abrogated in the presence of receptor-associated protein-1, an antagonist of the low-density lipoprotein receptor family, which also inhibited MT- and EmtinB-induced neurite outgrowth and survival. MT-mediated neurite outgrowth was furthermore inhibited by an anti-megalin serum. EmtinB-mediated inhibition of apoptosis occurred without a reduction of caspase-3 activity, but was associated with reduced expression of the pro-apoptotic B-cell leukemia/lymphoma-2 interacting member of cell death (Bim_S). Finally, evidence is provided that MT and EmtinB activate extracellular signal-regulated kinase, protein kinase B, and cAMP response element binding protein. Altogether, these results strongly suggest that MT and EmtinB induce their neuronal effects through direct binding to surface receptors belonging to the low-density lipoprotein receptor family, such as megalin and LRP, thereby activating signal transduction pathways resulting in neurite outgrowth and survival.     

Structural features specific to plant metallothioneins

The metallothionein (MT) superfamily combines a large variety of small cysteine-rich proteins from nearly all phyla of life that have the ability to coordinate various transition metal ions, including Zn^II, Cd^II, and Cu^I. The members of the plant MT family are characterized by great sequence diversity, requiring further subdivision into four subfamilies. Very peculiar and not well understood is the presence of rather long cysteine-free amino acid linkers between the cysteine-rich regions. In light of the distinct differences in sequence to MTs from other families, it seems obvious to assume that these differences will also be manifested on the structural level. This was already impressively demonstrated with the elucidation of the three-dimensional structure of the wheat E_c-1 MT, which revealed two metal cluster arrangements previously unprecedented for any MT. However, as this structure is so far the only one available for the plant MT family, other sources of information are in high demand. In this review the focus is thus set on any structural features known, deduced, or assumed for the plant MT proteins. This includes the determination of secondary structural elements by circular dichroism, IR, and Raman spectroscopy, the analysis of the influence of the long linker regions, and the evaluation of the spatial arrangement of the sequence separated cysteine-rich regions with the aid of, e.g., limited proteolytic digestion. In addition, special attention is paid to the contents of divalent metal ions as the metal ion to cysteine ratios are important for predicting and understanding possible metal–thiolate cluster structures.     

Exclusion of linkage between the human apolipoprotein B gene and abetalipoproteinemia

Abetalipoproteinemia (ABLP) is a rare autosomal recessive disease characterized by a lack of plasma apolipoprotein B (apo B). In this report, the hypothesis that ABLP is due to rare mutations in the apo B gene was tested. A total of eight ABLP families were studied. Apo B gene RFLPs were used to establish the haplotypes of the apo B alleles in family members. LOD score analysis was used to study the linkage between the apo B alleles and ABLP. These families were categorized arbitrarily as class I, II, III, or IV because of differences in the results derived from both haplotyping and LOD score analysis. In a class I family, affected siblings, who on the basis of the hypothesis would be expected to have the same apo B alleles, had different ones. LOD score analysis of this family gave an infinite negative number at a recombination fraction (theta) of zero. In two class II families, probands who were the result of consanguineous marriages and who, on the basis of the hypothesis, should be homozygotes for a defective apo B allele, were heterozygotes at this locus. The sum of the LOD scores from these two families was -1.7 at theta = 0. In one class III family, a parent was apparently homozygous for a particular apo B allele and yet not affected. This also contributed negatively to the LOD score. In four class IV families, disease inheritance was compatible with segregation of the apo B alleles. This, however, was not statistically significant (LOD score = 0.97 at theta = 0).(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalytically Inactive Protein Phosphatase 2A Can Bind to Polyomavirus Middle Tumor Antigen and Support Complex Formation with pp60c-src

Interaction between the heterodimeric form of protein phosphatase 2A (PP2A) and polyomavirus middle T antigen (MT) is required for the subsequent assembly of a transformation-competent MT complex. To investigate the role of PP2A catalytic activity in MT complex formation, we undertook a mutational analysis of the PP2A 36-kDa catalytic C subunit. Several residues likely to be involved in the dephosphorylation mechanism were identified and mutated. The resultant catalytically inactive C subunit mutants were then analyzed for their ability to associate with a cellular (B subunit) or a viral (MT) B-type subunit. Strikingly, while all of the inactive mutants were severely impaired in their interaction with B subunit, most of these mutants formed complexes with polyomavirus MT. These findings indicate a potential role for these catalytically important residues in complex formation with cellular B subunit, but not in complex formation with MT. Transformation-competent MT is known to associate with, and modulate the activity of, several cellular proteins, including pp60(c-src) family kinases. To determine whether association of MT with an active PP2A A-C heterodimer is necessary for subsequent association with pp60(c-src), catalytically inactive C subunits were examined for their ability to form complexes containing pp60(c-src) in MT-expressing cells. Two catalytically inactive C subunit mutants that efficiently formed complexes with MT also formed complexes that included an active pp60(c-src) kinase, demonstrating that PP2A activity is not essential in cis in MT complexes for subsequent pp60(c-src) association.     

Human endothelial gelatinases and angiogenesis

Endothelial cell invasion is an essential event during angiogenesis (formation of new blood vessels). The process involves the degradation of the basement membrane and the underlying interstitium. The matrix metalloproteinase (MMP) family is considered to be primarily responsible for matrix degradation. Two members of the family, gelatinase A and B play an important role in angiogenesis. This review outlines recent findings on their regulation in human endothelial cells. Latent gelatinase B is secreted from endothelial cells. This enzyme can also accumulate in the cytosol as an active enzyme, free of TIMP-1. In contrast, latent gelatinase A is constitutively secreted from the cells. Unlike other MMPs, gelatinase A activation occurs on the cell membrane and is mediated by MT1-MMP. A number of physiological activators have recently been described. These include thrombin and activated protein C, both of which activate gelatinase A independent of the MT1-MMP pathway. These new findings may lead to therapeutic interventions for the treatment of angiogenic-dependent diseases such as cancer and arthritis.     

Rates and relations of mitochondrial genome evolution across the Echinoidea,with special focus on the superfamily Odontophora

In order to better characterize the placement of genus Tripneustes, as a representative of the Toxopneustidae family within the broader sea urchin mitochondrial (MT) phylogeny, the complete MT genome of Tripneustes gratilla was generated and compared with all published echinoid MT genomes currently available on NCBI GenBank. The MT genome phylogeny supports the existence of the superfamily Odontophora (consisting of the families Strongylocentrotidae, Echinometridae, and Toxopneustidae). A relaxed molecular‐clock time calibration suggests a split between the three key Odontophore MT lineages occurred during the late Eocene/Oligocene. Major global oceanographic changes have been inferred during this time frame, potentially driving species diversification through environmental selection pressures. To test for signatures of selection acting on the mitochondria, the historical rate of gene evolution of individual MT genes was assessed through a branch‐site comparison of nonsynonymous to synonymous substitution ratios (ω). Models of positive selection and neutral evolution, as compared via a likelihood ratio test, show no evidence of strong historical positive selection on mitochondrial genes at the genesis of the Odontophora. However, while pairwise ω comparison revealed signatures of strong negative selection, relatively elevated ω values were observed within the Strongylocentrotus genus.     

三种被动式采集方法对甲虫收集效果的比较研究: 以香港城门样地为例

标本标准化采集是昆虫多样性研究的根本。昆虫种类繁多、习性复杂、分布广泛, 基于不同的研究目标, 昆虫学家会选用不同的采集方法。由于主动式采集方法存在较多干扰因素和重现性差等问题, 以飞行阻隔器(flight interception trap, FIT)、马氏网(Malaise trap, MT)和罐诱(pitfall trap, PT)为代表的被动式采集方法被广泛应用, 并在昆虫多样性研究中展现独特的优势。然而关于这些被动式采集方法的收集特点和采集效果等还缺乏系统性研究。本研究选取香港城门13个样点, 利用上述3种被动式采集方法共156个采集装备开展为期24天的鞘翅目昆虫采集工作, 并通过多样性指数分析、多度分析、体型与食性相关分析、相似性分析以及物种累积曲线分析评估了不同采集方法对甲虫的收集效果。本研究共采集甲虫6,380头, 涉及40科197种, 分析结果显示: (1)采用不同采集方法获得的物种数量和组成存在差异。从科级和种级的数量来看, FIT (36科, 149种) > MT (24科, 79种) > PT (17科, 60种); 在物种组成方面, FIT与PT之间、MT与PT之间区别较大, FIT与MT对应的物种相似度稍高于前两组。(2)多样性指数和物种多度分布分析显示: 丰富度指数为FIT > MT > PT, 优势度指数为FIT > PT > MT, 多样性指数为MT > FIT > PT, 均匀度指数为MT > PT > FIT。3种方法采集到的甲虫个体数为1头的种较多, 个体数超过1头的种在时间和空间方面的分布较广, 优势科的种类较少, 但其个体数占总个体数的比例较高。(3) FIT和PT均采集到了6类食性的甲虫, 其中藻食性的缨甲科甲虫仅见于FIT和PT采集方法。(4)物种累积曲线的结果表明3种采集方法效果均较好。3种采集方法各有特点, 但FIT采集的综合效果最优。FIT和MT两种方法的结合提升了采集甲虫的种类、食性和体型等方面的覆盖度, 更利于对甲虫多样性及类群与生态环境功能互作的研究。3种方法所收集到的甲虫存在一定差异, 因此可以针对不同研究目的选取适宜的采集方式。     

Absence of mechanical allodynia and Abeta-fiber sprouting after sciatic nerve injury in mice lacking membrane-type 5 matrix metalloproteinase

Matrix metalloproteinases (MMPs) are a family of endopeptidases that degrade extracellular matrix components. Membrane-type 5 MMP (MT5-MMP/MMP-24) was identified as neuron-specific, and is believed to contribute to neuronal circuit formation and plasticity. To elucidate its function in vivo, we have generated mice lacking MT5-MMP by gene targeting. MT5-MMP-deficient mice were born without obvious morphological abnormalities. No apparent histological defects were observed in the nervous system either. However, MT5-MMP-deficient mice did not develop neuropathic pain with mechanical allodynia after sciatic nerve injury, though responses to acute noxious stimuli were normal. Neuropathic pain induced by peripheral nerve lesions is known to accompany structural reorganization of the nervous system. Intraneural injection of cholera toxin B subunit, a transganglionic tracer, into the injured sciatic nerve of wild-type mice revealed that the myelinated Abeta-fiber primary afferents sprouted from laminae III-VI of the dorsal horn of the spinal cord and invaded lamina II. However, no such sprouting and invasion of Abeta-fibers were observed in MT5-MMP-deficient mice. These findings suggest that MT5-MMP is essential for the development of mechanical allodynia and plays an important role in neuronal plasticity in this mouse model.