The nuclear F-actin interactome of Xenopus oocytes reveals an actin-bundling kinesin that is essential for meiotic cytokinesis

PubMed Central

Samwer, Matthias; Dehne, Heinz-Jürgen; Spira, Felix; Kollmar, Martin; Gerlich, Daniel W; Urlaub, Henning; Görlich, Dirk

2013-01-01

Nuclei of Xenopus laevis oocytes grow 100 000-fold larger in volume than a typical somatic nucleus and require an unusual intranuclear F-actin scaffold for mechanical stability. We now developed a method for mapping F-actin interactomes and identified a comprehensive set of F-actin binders from the oocyte nuclei. Unexpectedly, the most prominent interactor was a novel kinesin termed NabKin (Nuclear and meiotic actin-bundling Kinesin). NabKin not only binds microtubules but also F-actin structures, such as the intranuclear actin bundles in prophase and the contractile actomyosin ring during cytokinesis. The interaction between NabKin and F-actin is negatively regulated by Importin-β and is responsive to spatial information provided by RanGTP. Disconnecting NabKin from F-actin during meiosis caused cytokinesis failure and egg polyploidy. We also found actin-bundling activity in Nabkin's somatic paralogue KIF14, which was previously shown to be essential for somatic cell division. Our data are consistent with the notion that NabKin/KIF14 directly link microtubules with F-actin and that such link is essential for cytokinesis. PMID:23727888

Kinesin-5, a mitotic microtubule-associated motor protein, modulates neuronal migration

PubMed Central

Falnikar, Aditi; Tole, Shubha; Baas, Peter W.

2011-01-01

Kinesin-5 (also called Eg5 or kif11) is a homotetrameric motor protein that functions by modulating microtubule (MT)–MT interactions. In the case of mitosis, kinesin-5 slows the rate of separation of the half-spindles. In the case of the axon, kinesin-5 limits the frequency of transport of short MTs, and also limits the rate of axonal growth. Here we show that experimental inhibition of kinesin-5 in cultured migratory neurons results in a faster but more randomly moving neuron with a shorter leading process. As is the case with axons of stationary neurons, short MT transport frequency is notably enhanced in the leading process of the migratory neuron when kinesin-5 is inhibited. Conversely, overexpression of kinesin-5, both in culture and in developing cerebral cortex, causes migration to slow and even cease. Regions of anti-parallel MT organization behind the centrosome were shown to be especially rich in kinesin-5, implicating these regions as potential sites where kinesin-5 forces may be especially relevant. We posit that kinesin-5 acts as a “brake” on MT–MT interactions that modulates the advance of the entire MT apparatus. In so doing, kinesin-5 regulates the rate and directionality of neuronal migration and possibly the cessation of migration when the neuron reaches its destination. PMID:21411631

The spindle kinesin-like protein HsEg5 is an autoantigen in systemic lupus erythematosus.

PubMed

Whitehead, C M; Winkfein, R J; Fritzler, M J; Rattner, J B

1996-10-01

Autoantibodies directed against the mitotic spindle apparatus (MSA) have been shown to target an antigen referred to as NuMA (nuclear mitotic apparatus). In this study, we identified a second MSA antigen as the spindle kinesin-like protein HsEg5. We studied the frequency of antibodies to HsEg5 in human sera that demonstrate the MSA pattern of staining, the frequency of autoantibodies to HsEg5 in patients with systemic lupus erythematosus (SLE), and the clinical features of patients with antibodies to HsEg5. A prototype serum from an SLE patient was used to isolate a 4.8-kilobase complementary DNA (cDNA) from a HeLa cDNA library. Western blot, immunoprecipitation, and sequence analysis revealed that the antigen was an approximately 130-kd protein, HsEg5. The frequency of autoantibodies to recombinant HsEg5 in 51 sera that demonstrated an MSA pattern of staining on HEp-2 and HeLa cells was detected by immunoblotting 2 constructs of the cDNA. The clinical features of patients with antibodies directed against HsEg5 was obtained by retrospective chart review. The antigen responsible for the MSA-35 pattern was identified as the human kinesin-like protein HsEg5. Seven of 51 sera (14%) that demonstrated an MSA pattern of staining reacted with recombinant HsEg5. Six of 7 of the HsEg5-positive patients (86%) had SLE, and 1 had Sjögren's syndrome. The indirect immunofluorescent staining pattern of sera that reacted with HsEg5 could be distinguished from the other sera that reacted with NuMA. In an unselected cohort of 52 SLE patients, 3 (6%) had autoantibodies reactive with the recombinant HsEg5. Autoantibodies to MSA fall into 2 major classes: those reactive with NuMA and those reactive with HsEg5. Autoantibodies to HsEg5 are found in a lower frequency than NuMA in sera that demonstrate the MSA pattern of staining and appear to be specifically associated with SLE. HsEg5 can be distinguished from NuMA by indirect immunofluorescence and Western blotting.

Detection of the quantity of kinesin and microgravity-sensitive kinesin genes in rat bone marrow stromal cells grown in a simulated microgravity environment

NASA Astrophysics Data System (ADS)

Ni, Chengzhi; Wang, Chunyan; Li, Yuan; Li, Yinghui; Dai, Zhongquan; Zhao, Dongming; Sun, Hongyi; Wu, Bin

2011-06-01

Kinesin and kinesin-like proteins (KLPs) constitute a superfamily of microtubule motor proteins found in all eukaryotic organisms. Members of the kinesin superfamily are known to play important roles in many fundamental cellular and developmental processes. To date, few published studies have reported on the effects of microgravity on kinesin expression. In this paper, we describe the expression pattern and microgravity-sensitive genes of kinesin in rat bone marrow stromal cells cultured in a ground-based rotating bioreactor. The quantity of kinesin under the clinorotation condition was examined by immunoblot analysis with anti-kinesin. Furthermore, the distribution of kinesin at various times during clinorotation was determined by dual immunostaining, using anti-kinesin monoclonal antibody or anti-β-tubulin monoclonal antibody. In terms of kinesin quantity, we found that the ratios of the amounts of clinorotated/stationary KLPs decreased from clinorotation day 5 to day 10, although it increased on days 2 and 3. Immunofluorescence analysis revealed that kinesin in the nucleus was the first to be affected by simulated microgravity, following the kinesin at the periphery that was affected at various times during clinorotation. Real-time RT-PCR analysis of kinesin mRNA expression was performed and led to the identification of 3 microgravity-sensitive kinesin genes: KIF9, KIFC1, and KIF21A. Our results suggest that kinesin has a distinct expression pattern, and the identification of microgravity-sensitive kinesin genes offers insight into fundamental cell biology.

Correlation Imaging Reveals Specific Crowding Dynamics of Kinesin Motor Proteins

NASA Astrophysics Data System (ADS)

Miedema, Daniël M.; Kushwaha, Vandana S.; Denisov, Dmitry V.; Acar, Seyda; Nienhuis, Bernard; Peterman, Erwin J. G.; Schall, Peter

2017-10-01

Molecular motor proteins fulfill the critical function of transporting organelles and other building blocks along the biopolymer network of the cell's cytoskeleton, but crowding effects are believed to crucially affect this motor-driven transport due to motor interactions. Physical transport models, like the paradigmatic, totally asymmetric simple exclusion process (TASEP), have been used to predict these crowding effects based on simple exclusion interactions, but verifying them in experiments remains challenging. Here, we introduce a correlation imaging technique to precisely measure the motor density, velocity, and run length along filaments under crowding conditions, enabling us to elucidate the physical nature of crowding and test TASEP model predictions. Using the kinesin motor proteins kinesin-1 and OSM-3, we identify crowding effects in qualitative agreement with TASEP predictions, and we achieve excellent quantitative agreement by extending the model with motor-specific interaction ranges and crowding-dependent detachment probabilities. These results confirm the applicability of basic nonequilibrium models to the intracellular transport and highlight motor-specific strategies to deal with crowding.

Mutation of Rice BC12/GDD1, Which Encodes a Kinesin-Like Protein That Binds to a GA Biosynthesis Gene Promoter, Leads to Dwarfism with Impaired Cell Elongation[W][OA

PubMed Central

Li, Juan; Jiang, Jiafu; Qian, Qian; Xu, Yunyuan; Zhang, Cui; Xiao, Jun; Du, Cheng; Luo, Wei; Zou, Guoxing; Chen, Mingluan; Huang, Yunqing; Feng, Yuqi; Cheng, Zhukuan; Yuan, Ming; Chong, Kang

2011-01-01

The kinesins are a family of microtubule-based motor proteins that move directionally along microtubules and are involved in many crucial cellular processes, including cell elongation in plants. Less is known about kinesins directly regulating gene transcription to affect cellular physiological processes. Here, we describe a rice (Oryza sativa) mutant, gibberellin-deficient dwarf1 (gdd1), that has a phenotype of greatly reduced length of root, stems, spikes, and seeds. This reduced length is due to decreased cell elongation and can be rescued by exogenous gibberellic acid (GA3) treatment. GDD1 was cloned by a map-based approach, was expressed constitutively, and was found to encode the kinesin-like protein BRITTLE CULM12 (BC12). Microtubule cosedimentation assays revealed that BC12/GDD1 bound to microtubules in an ATP-dependent manner. Whole-genome microarray analysis revealed the expression of ent-kaurene oxidase (KO2), which encodes an enzyme involved in GA biosynthesis, was downregulated in gdd1. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that GDD1 bound to the element ACCAACTTGAA in the KO2 promoter. In addition, GDD1 was shown to have transactivation activity. The level of endogenous GAs was reduced in gdd1, and the reorganization of cortical microtubules was altered. Therefore, BC12/GDD1, a kinesin-like protein with transcription regulation activity, mediates cell elongation by regulating the GA biosynthesis pathway in rice. PMID:21325138

Bidirectional motility of kinesin-5 motor proteins: structural determinants, cumulative functions and physiological roles.

PubMed

Singh, Sudhir Kumar; Pandey, Himanshu; Al-Bassam, Jawdat; Gheber, Larisa

2018-05-01

Mitotic kinesin-5 bipolar motor proteins perform essential functions in mitotic spindle dynamics by crosslinking and sliding antiparallel microtubules (MTs) apart within the mitotic spindle. Two recent studies have indicated that single molecules of Cin8, the Saccharomyces cerevisiae kinesin-5 homolog, are minus end-directed when moving on single MTs, yet switch directionality under certain experimental conditions (Gerson-Gurwitz et al., EMBO J 30:4942-4954, 2011; Roostalu et al., Science 332:94-99, 2011). This finding was unexpected since the Cin8 catalytic motor domain is located at the N-terminus of the protein, and such kinesins have been previously thought to be exclusively plus end-directed. In addition, the essential intracellular functions of kinesin-5 motors in separating spindle poles during mitosis can only be accomplished by plus end-directed motility during antiparallel sliding of the spindle MTs. Thus, the mechanism and possible physiological role of the minus end-directed motility of kinesin-5 motors remain unclear. Experimental and theoretical studies from several laboratories in recent years have identified additional kinesin-5 motors that are bidirectional, revealed structural determinants that regulate directionality, examined the possible mechanisms involved and have proposed physiological roles for the minus end-directed motility of kinesin-5 motors. Here, we summarize our current understanding of the remarkable ability of certain kinesin-5 motors to switch directionality when moving along MTs.

Kinesin molecular motors: Transport pathways, receptors, and human disease

NASA Astrophysics Data System (ADS)

Goldstein, Lawrence S. B.

2001-06-01

Kinesin molecular motor proteins are responsible for many of the major microtubule-dependent transport pathways in neuronal and non-neuronal cells. Elucidating the transport pathways mediated by kinesins, the identity of the cargoes moved, and the nature of the proteins that link kinesin motors to cargoes are areas of intense investigation. Kinesin-II recently was found to be required for transport in motile and nonmotile cilia and flagella where it is essential for proper left-right determination in mammalian development, sensory function in ciliated neurons, and opsin transport and viability in photoreceptors. Thus, these pathways and proteins may be prominent contributors to several human diseases including ciliary dyskinesias, situs inversus, and retinitis pigmentosa. Kinesin-I is needed to move many different types of cargoes in neuronal axons. Two candidates for receptor proteins that attach kinesin-I to vesicular cargoes were recently found. One candidate, sunday driver, is proposed to both link kinesin-I to an unknown vesicular cargo and to bind and organize the mitogen-activated protein kinase components of a c-Jun N-terminal kinase signaling module. A second candidate, amyloid precursor protein, is proposed to link kinesin-I to a different, also unknown, class of axonal vesicles. The finding of a possible functional interaction between kinesin-I and amyloid precursor protein may implicate kinesin-I based transport in the development of Alzheimer's disease.

A novel kinesin-like protein, KIF1Bbeta3 is involved in the movement of lysosomes to the cell periphery in non-neuronal cells.

PubMed

Matsushita, Masafumi; Tanaka, Shingo; Nakamura, Norihiro; Inoue, Hiroki; Kanazawa, Hiroshi

2004-03-01

The kinesin superfamily protein, KIF1Bbeta, a splice variant of KIF1B, is involved in the transport of synaptic vesicles in neuronal cells, and is also expressed in various non-neuronal tissues. To elucidate the functions of KIF1Bbeta in non-neuronal cells, we analyzed the intracellular localization of KIF1Bbeta and characterized its isoform expression profile. In COS-7 cells, KIF1B colocalized with lysosomal markers and expression of a mutant form of KIF1Bbeta, lacking the motor domain, impaired the intracellular distribution of lysosomes. A novel isoform of the kinesin-like protein, KIF1Bbeta3, was identified in rat and simian kidney. It lacks the 5th exon of the KIF1Bbeta-specific tail region. Overexpression of KIF1Bbeta3 induced the translocation of lysosomes to the cell periphery. However, overexpression of KIF1Bbeta3-Q98L, which harbors a pathogenic mutation associated with a familial neuropathy, Charcot-Marie-Tooth disease type 2 A, resulted in the abnormal perinuclear clustering of lysosomes. These results indicate that KIF1Bbeta3 is involved in the translocation of lysosomes from perinuclear regions to the cell periphery.

Functional Analysis of Human Microtubule-based Motor Proteins, the Kinesins and Dyneins, in Mitosis/Cytokinesis Using RNA InterferenceD⃞V⃞

PubMed Central

Zhu, Changjun; Zhao, Jian; Bibikova, Marina; Leverson, Joel D.; Bossy-Wetzel, Ella; Fan, Jian-Bing; Abraham, Robert T.; Jiang, Wei

2005-01-01

Microtubule (MT)-based motor proteins, kinesins and dyneins, play important roles in multiple cellular processes including cell division. In this study, we describe the generation and use of an Escherichia coli RNase III-prepared human kinesin/dynein esiRNA library to systematically analyze the functions of all human kinesin/dynein MT motor proteins. Our results indicate that at least 12 kinesins are involved in mitosis and cytokinesis. Eg5 (a member of the kinesin-5 family), Kif2A (a member of the kinesin-13 family), and KifC1 (a member of the kinesin-14 family) are crucial for spindle formation; KifC1, MCAK (a member of the kinesin-13 family), CENP-E (a member of the kinesin-7 family), Kif14 (a member of the kinesin-3 family), Kif18 (a member of the kinesin-8 family), and Kid (a member of the kinesin-10 family) are required for chromosome congression and alignment; Kif4A and Kif4B (members of the kinesin-4 family) have roles in anaphase spindle dynamics; and Kif4A, Kif4B, MKLP1, and MKLP2 (members of the kinesin-6 family) are essential for cytokinesis. Using immunofluorescence analysis, time-lapse microscopy, and rescue experiments, we investigate the roles of these 12 kinesins in detail. PMID:15843429

EBF proteins participate in transcriptional regulation of Xenopus muscle development.

PubMed

Green, Yangsook Song; Vetter, Monica L

2011-10-01

EBF proteins have diverse functions in the development of multiple lineages, including neurons, B cells and adipocytes. During Drosophila muscle development EBF proteins are expressed in muscle progenitors and are required for muscle cell differentiation, but there is no known function of EBF proteins in vertebrate muscle development. In this study, we examine the expression of ebf genes in Xenopus muscle tissue and show that EBF activity is necessary for aspects of Xenopus skeletal muscle development, including somite organization, migration of hypaxial muscle anlagen toward the ventral abdomen, and development of jaw muscle. From a microarray screen, we have identified multiple candidate targets of EBF activity with known roles in muscle development. The candidate targets we have verified are MYOD, MYF5, M-Cadherin and SEB-4. In vivo overexpression of the ebf2 and ebf3 genes leads to ectopic expression of these candidate targets, and knockdown of EBF activity causes downregulation of the endogenous expression of the candidate targets. Furthermore, we found that MYOD and MYF5 are likely to be direct targets. Finally we show that MYOD can upregulate the expression of ebf genes, indicating the presence of a positive feedback loop between EBF and MYOD that we find to be important for maintenance of MYOD expression in Xenopus. These results suggest that EBF activity is important for both stabilizing commitment and driving aspects of differentiation in Xenopus muscle cells. Copyright © 2010 Elsevier Inc. All rights reserved.

The transport of Staufen2-containing ribonucleoprotein complexes involves kinesin motor protein and is modulated by mitogen-activated protein kinase pathway.

PubMed

Jeong, Ji-Hye; Nam, Yeon-Ju; Kim, Seok-Yong; Kim, Eung-Gook; Jeong, Jooyoung; Kim, Hyong Kyu

2007-09-01

There is increasing evidence showing that mRNA is transported to the neuronal dendrites in ribonucleoprotein (RNP) complexes or RNA granules, which are aggregates of mRNA, rRNA, ribosomal proteins, and RNA-binding proteins. In these RNP complexes, Staufen, a double-stranded RNA-binding protein, is believed to be a core component that plays a key role in the dendritic mRNA transport. This study investigated the molecular mechanisms of the dendritic mRNA transport using green fluorescent protein-tagged Staufen2 produced employing a Sindbis viral expression system. The kinesin heavy chain was found to be associated with Staufen2. The inhibition of kinesin resulted in a significant decrease in the level of dendritic transport of the Staufen2-containing RNP complexes in neurons under non-stimulating or stimulating conditions. This suggests that the dendritic transport of the Staufen2-containing RNP complexes use kinesin as a motor protein. A mitogen-activated protein kinase inhibitor, PD98059, inhibited the activity-induced increase in the amount of both the Staufen2-containing RNP complexes and Ca(2+)/calmodulin-dependent protein kinase II alpha-subunit mRNA in the distal dendrites of cultured hippocampal neurons. Overall, these results suggest that dendritic mRNA transport is mediated via the Staufen2 and kinesin motor proteins and might be modulated by the neuronal activity and mitogen-activated protein kinase pathway.

Tumour Suppressor Adenomatous Polyposis Coli (APC) localisation is regulated by both Kinesin-1 and Kinesin-2.

PubMed

Ruane, Peter T; Gumy, Laura F; Bola, Becky; Anderson, Beverley; Wozniak, Marcin J; Hoogenraad, Casper C; Allan, Victoria J

2016-06-07

Microtubules and their associated proteins (MAPs) underpin the polarity of specialised cells. Adenomatous polyposis coli (APC) is one such MAP with a multifunctional agenda that requires precise intracellular localisations. Although APC has been found to associate with kinesin-2 subfamily members, the exact mechanism for the peripheral localization of APC remains unclear. Here we show that the heavy chain of kinesin-1 directly interacts with the APC C-terminus, contributing to the peripheral localisation of APC in fibroblasts. In rat hippocampal neurons the kinesin-1 binding domain of APC is required for its axon tip enrichment. Moreover, we demonstrate that APC requires interactions with both kinesin-2 and kinesin-1 for this localisation. Underlining the importance of the kinesin-1 association, neurons expressing APC lacking kinesin-1-binding domain have shorter axons. The identification of this novel kinesin-1-APC interaction highlights the complexity and significance of APC localisation in neurons.

Tumour Suppressor Adenomatous Polyposis Coli (APC) localisation is regulated by both Kinesin-1 and Kinesin-2

PubMed Central

Ruane, Peter T.; Gumy, Laura F.; Bola, Becky; Anderson, Beverley; Wozniak, Marcin J.; Hoogenraad, Casper C.; Allan, Victoria J.

2016-01-01

Microtubules and their associated proteins (MAPs) underpin the polarity of specialised cells. Adenomatous polyposis coli (APC) is one such MAP with a multifunctional agenda that requires precise intracellular localisations. Although APC has been found to associate with kinesin-2 subfamily members, the exact mechanism for the peripheral localization of APC remains unclear. Here we show that the heavy chain of kinesin-1 directly interacts with the APC C-terminus, contributing to the peripheral localisation of APC in fibroblasts. In rat hippocampal neurons the kinesin-1 binding domain of APC is required for its axon tip enrichment. Moreover, we demonstrate that APC requires interactions with both kinesin-2 and kinesin-1 for this localisation. Underlining the importance of the kinesin-1 association, neurons expressing APC lacking kinesin-1-binding domain have shorter axons. The identification of this novel kinesin-1-APC interaction highlights the complexity and significance of APC localisation in neurons. PMID:27272132

Release of kinesin from vesicles by hsc70 and regulation of fast axonal transport

NASA Technical Reports Server (NTRS)

Tsai, M. Y.; Morfini, G.; Szebenyi, G.; Brady, S. T.

2000-01-01

The nature of kinesin interactions with membrane-bound organelles and mechanisms for regulation of kinesin-based motility have both been surprisingly difficult to define. Most kinesin is recovered in supernatants with standard protocols for purification of motor proteins, but kinesin recovered on membrane-bound organelles is tightly bound. Partitioning of kinesin between vesicle and cytosolic fractions is highly sensitive to buffer composition. Addition of either N-ethylmaleimide or EDTA to homogenization buffers significantly increased the fraction of kinesin bound to organelles. Given that an antibody against kinesin light chain tandem repeats also releases kinesin from vesicles, these observations indicated that specific cytoplasmic factors may regulate kinesin release from membranes. Kinesin light tandem repeats contain DnaJ-like motifs, so the effects of hsp70 chaperones were evaluated. Hsc70 released kinesin from vesicles in an MgATP-dependent and N-ethylmaleimide-sensitive manner. Recombinant kinesin light chains inhibited kinesin release by hsc70 and stimulated the hsc70 ATPase. Hsc70 actions may provide a mechanism to regulate kinesin function by releasing kinesin from cargo in specific subcellular domains, thereby effecting delivery of axonally transported materials.

Discovery of a novel inhibitor of kinesin-like protein KIFC1.

PubMed

Zhang, Wei; Zhai, Ling; Wang, Yimin; Boohaker, Rebecca J; Lu, Wenyan; Gupta, Vandana V; Padmalayam, Indira; Bostwick, Robert J; White, E Lucile; Ross, Larry J; Maddry, Joseph; Ananthan, Subramaniam; Augelli-Szafran, Corinne E; Suto, Mark J; Xu, Bo; Li, Rongbao; Li, Yonghe

2016-04-15

Historically, drugs used in the treatment of cancers also tend to cause damage to healthy cells while affecting cancer cells. Therefore, the identification of novel agents that act specifically against cancer cells remains a high priority in the search for new therapies. In contrast with normal cells, most cancer cells contain multiple centrosomes which are associated with genome instability and tumorigenesis. Cancer cells can avoid multipolar mitosis, which can cause cell death, by clustering the extra centrosomes into two spindle poles, thereby enabling bipolar division. Kinesin-like protein KIFC1 plays a critical role in centrosome clustering in cancer cells, but is not essential for normal cells. Therefore, targeting KIFC1 may provide novel insight into selective killing of cancer cells. In the present study, we identified a small-molecule KIFC1 inhibitor, SR31527, which inhibited microtubule (MT)-stimulated KIFC1 ATPase activity with an IC50 value of 6.6 μM. By using bio layer interferometry technology, we further demonstrated that SR31527 bound directly to KIFC1 with high affinity (Kd=25.4 nM). Our results from computational modelling and saturation-transfer difference (STD)-NMR experiments suggest that SR31527 bound to a novel allosteric site of KIFC1 that appears suitable for developing selective inhibitors of KIFC1. Importantly, SR31527 prevented bipolar clustering of extra centrosomes in triple negative breast cancer (TNBC) cells and significantly reduced TNBC cell colony formation and viability, but was less toxic to normal fibroblasts. Therefore, SR31527 provides a valuable tool for studying the biological function of KIFC1 and serves as a potential lead for the development of novel therapeutic agents for breast cancer treatment. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Three new members of the RNP protein family in Xenopus.

PubMed Central

Good, P J; Rebbert, M L; Dawid, I B

1993-01-01

Many RNP proteins contain one or more copies of the RNA recognition motif (RRM) and are thought to be involved in cellular RNA metabolism. We have previously characterized in Xenopus a nervous system specific gene, nrp1, that is more similar to the hnRNP A/B proteins than to other known proteins (K. Richter, P. J. Good, and I. B. Dawid (1990), New Biol. 2, 556-565). PCR amplification with degenerate primers was used to identify additional cDNAs encoding two RRMs in Xenopus. Three previously uncharacterized genes were identified. Two genes encode hnRNP A/B proteins with two RRMs and a glycine-rich domain. One of these is the Xenopus homolog of the human A2/B1 gene; the other, named hnRNP A3, is similar to both the A1 and A2 hnRNP genes. The Xenopus hnRNP A1, A2 and A3 genes are expressed throughout development and in all adult tissues. Multiple protein isoforms for the hnRNP A2 gene are predicted that differ by the insertion of short peptide sequences in the glycine-rich domain. The third newly isolated gene, named xrp1, encodes a protein that is related by sequence to the nrp1 protein but is expressed ubiquitously. Despite the similarity to nuclear RNP proteins, both the nrp1 and xrp1 proteins are localized to the cytoplasm in the Xenopus oocyte. The xrp1 gene may have a function in all cells that is similar to that executed by nrp1 specifically within the nervous system. Images PMID:8451200

Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization.

PubMed

Roux, Kyle J; Crisp, Melissa L; Liu, Qian; Kim, Daein; Kozlov, Serguei; Stewart, Colin L; Burke, Brian

2009-02-17

Nucleocytoplasmic coupling is mediated by outer nuclear membrane (ONM) nesprin proteins and inner nuclear membrane Sun proteins. Interactions spanning the perinuclear space create nesprin-Sun complexes connecting the cytoskeleton to nuclear components. A search for proteins displaying a conserved C-terminal sequence present in nesprins 1-3 identified nesprin 4 (Nesp4), a new member of this family. Nesp4 is a kinesin-1-binding protein that displays Sun-dependent localization to the ONM. Expression of Nesp4 is associated with dramatic changes in cellular organization involving relocation of the centrosome and Golgi apparatus relative to the nucleus. These effects can be accounted for entirely by Nesp4's kinesin-binding function. The implication is that Nesp4 may contribute to microtubule-dependent nuclear positioning.

A Chimeric Kinesin-1 Head/Kinesin-5 Tail Motor Switches between Diffusive and Processive Motility

PubMed Central

Thiede, Christina; Lakämper, Stefan; Wessel, Alok D.; Kramer, Stefanie; Schmidt, Christoph F.

2013-01-01

Homotetrameric kinesin-5 motors are essential for chromosome separation and assembly of the mitotic spindle. These kinesins bind between two microtubules (MTs) and slide them apart, toward the spindle poles. This process must be tightly regulated in mitosis. In in vitro assays, Eg5 moves diffusively on single MTs and switches to a directed mode between MTs. How allosteric communication between opposing motor domains works remains unclear, but kinesin-5 tail domains may be involved. Here we present a single-molecule fluorescence study of a tetrameric kinesin-1 head/kinesin-5 tail chimera, DK4mer. This motor exhibited fast processive motility on single MTs interrupted by pauses. Like Eg5, DK4mer diffused along MTs with ADP, and slid antiparallel MTs apart with ATP. In contrast to Eg5, diffusive and processive periods were clearly distinguishable. This allowed us to measure transition rates among states and for unbinding as a function of buffer ionic strength. These data, together with results from controls using tail-less dimers, indicate that there are two modes of interaction with MTs, separated by an energy barrier. This result suggests a scheme of motor regulation that involves switching between two bound states, possibly allosterically controlled by the opposing tetramer end. Such a scheme is likely to be relevant for the regulation of native kinesin-5 motors. PMID:23442865

Participation of Xenopus Elr-type Proteins in Vegetal mRNA Localization during Oogenesis*

PubMed Central

Arthur, Patrick K.; Claussen, Maike; Koch, Susanne; Tarbashevich, Katsiaryna; Jahn, Olaf; Pieler, Tomas

2009-01-01

Directional transport of specific mRNAs is of primary biological relevance. In Xenopus oocytes, mRNA localization to the vegetal pole is important for germ layer formation and germ cell development. Using a biochemical approach, we identified Xenopus Elr-type proteins, homologs of the Hu/ELAV proteins, as novel components of the vegetal mRNA localization machinery. They bind specifically to the localization elements of several different vegetally localizing Xenopus mRNAs, and they are part of one RNP together with other localization proteins, such as Vg1RBP and XStaufen 1. Blocking Elr-type protein binding by either localization element mutagenesis or antisense morpholino oligonucleotide-mediated masking of their target RNA structures, as well as overexpression of wild type and mutant ElrB proteins, interferes with vegetal localization in Xenopus oocytes. PMID:19458392

Kinesin 1 regulates cilia length through an interaction with the Bardet-Biedl syndrome related protein CCDC28B.

PubMed

Novas, Rossina; Cardenas-Rodriguez, Magdalena; Lepanto, Paola; Fabregat, Matías; Rodao, Magela; Fariello, María Inés; Ramos, Mauricio; Davison, Camila; Casanova, Gabriela; Alfaya, Lucía; Lecumberry, Federico; González-Sapienza, Gualberto; Irigoín, Florencia; Badano, Jose L

2018-02-14

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal degeneration, obesity, polydactyly, renal disease and mental retardation. CCDC28B is a BBS-associated protein that we have previously shown plays a role in cilia length regulation whereby its depletion results in shortened cilia both in cells and Danio rerio (zebrafish). At least part of that role is achieved by its interaction with the mTORC2 component SIN1, but the mechanistic details of this interaction and/or additional functions that CCDC28B might play in the context of cilia remain poorly understood. Here we uncover a novel interaction between CCDC28B and the kinesin 1 molecular motor that is relevant to cilia. CCDC28B interacts with kinesin light chain 1 (KLC1) and the heavy chain KIF5B. Notably, depletion of these kinesin 1 components results in abnormally elongated cilia. Furthermore, through genetic interaction studies we demonstrate that kinesin 1 regulates ciliogenesis through CCDC28B. We show that kinesin 1 regulates the subcellular distribution of CCDC28B, unexpectedly, inhibiting its nuclear accumulation, and a ccdc28b mutant missing a nuclear localization motif fails to rescue the phenotype in zebrafish morphant embryos. Therefore, we uncover a previously unknown role of kinesin 1 in cilia length regulation that relies on the BBS related protein CCDC28B.

Calculating the Degradation Rate of Individual Proteins Using Xenopus Extract Systems.

PubMed

McDowell, Gary S; Philpott, Anna

2018-05-16

The Xenopus extract system has been used extensively as a simple, quick, and robust method for assessing the stability of proteins against proteasomal degradation. In this protocol, methods are provided for assessing the half-life of in vitro translated radiolabeled proteins using Xenopus egg or embryo extracts. © 2019 Cold Spring Harbor Laboratory Press.

Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization

PubMed Central

Roux, Kyle J.; Crisp, Melissa L.; Liu, Qian; Kim, Daein; Kozlov, Serguei; Stewart, Colin L.; Burke, Brian

2009-01-01

Nucleocytoplasmic coupling is mediated by outer nuclear membrane (ONM) nesprin proteins and inner nuclear membrane Sun proteins. Interactions spanning the perinuclear space create nesprin–Sun complexes connecting the cytoskeleton to nuclear components. A search for proteins displaying a conserved C-terminal sequence present in nesprins 1–3 identified nesprin 4 (Nesp4), a new member of this family. Nesp4 is a kinesin-1-binding protein that displays Sun-dependent localization to the ONM. Expression of Nesp4 is associated with dramatic changes in cellular organization involving relocation of the centrosome and Golgi apparatus relative to the nucleus. These effects can be accounted for entirely by Nesp4's kinesin-binding function. The implication is that Nesp4 may contribute to microtubule-dependent nuclear positioning. PMID:19164528

A Mutation in γ-Tubulin Alters Microtubule Dynamics and Organization and Is Synthetically Lethal with the Kinesin-like Protein Pkl1pV⃞

PubMed Central

Paluh, Janet L.; Nogales, Eva; Oakley, Berl R.; McDonald, Kent; Pidoux, Alison L.; Cande, W. Z.

2000-01-01

Mitotic segregation of chromosomes requires spindle pole functions for microtubule nucleation, minus end organization, and regulation of dynamics. γ-Tubulin is essential for nucleation, and we now extend its role to these latter processes. We have characterized a mutation in γ-tubulin that results in cold-sensitive mitotic arrest with an elongated bipolar spindle but impaired anaphase A. At 30°C cytoplasmic microtubule arrays are abnormal and bundle into single larger arrays. Three-dimensional time-lapse video microscopy reveals that microtubule dynamics are altered. Localization of the mutant γ-tubulin is like the wild-type protein. Prediction of γ-tubulin structure indicates that non-α/β-tubulin protein–protein interactions could be affected. The kinesin-like protein (klp) Pkl1p localizes to the spindle poles and spindle and is essential for viability of the γ-tubulin mutant and in multicopy for normal cell morphology at 30°C. Localization and function of Pkl1p in the mutant appear unaltered, consistent with a redundant function for this protein in wild type. Our data indicate a broader role for γ-tubulin at spindle poles in regulating aspects of microtubule dynamics and organization. We propose that Pkl1p rescues an impaired function of γ-tubulin that involves non-tubulin protein–protein interactions, presumably with a second motor, MAP, or MTOC component. PMID:10749926

Crystal structure of the Candida albicans Kar3 kinesin motor domain fused to maltose-binding protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Delorme, Caroline; Joshi, Monika; Allingham, John S., E-mail: allinghj@queensu.ca

2012-11-30

Highlights: Black-Right-Pointing-Pointer The Candida albicans Kar3 motor domain structure was solved as a maltose-binding protein fusion. Black-Right-Pointing-Pointer The electrostatic surface and part of the ATPase pocket of the motor domain differs markedly from other kinesins. Black-Right-Pointing-Pointer The MBP-Kar3 interface highlights a new site for intramolecular or intermolecular interactions. -- Abstract: In the human fungal pathogen Candida albicans, the Kinesin-14 motor protein Kar3 (CaKar3) is critical for normal mitotic division, nuclear fusion during mating, and morphogenic transition from the commensal yeast form to the virulent hyphal form. As a first step towards detailed characterization of this motor of potential medical significance,more » we have crystallized and determined the X-ray structure of the motor domain of CaKar3 as a maltose-binding protein (MBP) fusion. The structure shows strong conservation of overall motor domain topology to other Kar3 kinesins, but with some prominent differences in one of the motifs that compose the nucleotide-binding pocket and the surface charge distribution. The MBP and Kar3 modules are arranged such that MBP interacts with the Kar3 motor domain core at the same site where the neck linker of conventional kinesins docks during the 'ATP state' of the mechanochemical cycle. This site differs from the Kar3 neck-core interface in the recent structure of the ScKar3Vik1 heterodimer. The position of MBP is also completely distinct from the Vik1 subunit in this complex. This may suggest that the site of MBP interaction on the CaKar3 motor domain provides an interface for the neck, or perhaps a partner subunit, at an intermediate state of its motile cycle that has not yet been observed for Kinesin-14 motors.« less

Casein Kinase 2 Reverses Tail-Independent Inactivation of Kinesin-1

NASA Astrophysics Data System (ADS)

Xu, Jing

2013-03-01

Kinesin-1 is a plus-end microtubule-based motor, and defects in kinesin-based transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a head-tail interaction, but is believed to be active otherwise. Here we report a tail-independent inactivation of kinesin, reversible by the disease-relevant signalling protein, casein kinase 2 (CK2). The majority of initially active kinesin (native or tail-less) loses its ability to interact with microtubules in vitro, and CK2 reverses this inactivation (approximately fourfold) without altering kinesin's single motor properties. This activation pathway does not require motor phosphorylation, and is independent of head-tail auto-inhibition. In cultured mammalian cells, reducing CK2 expression, but not its kinase activity, decreases the force required to stall lipid droplet transport, consistent with a decreased number of active kinesin motors. Our results (Nat. Commun., 3:754, 2012) provide the first direct evidence of a protein kinase upregulating kinesin-based transport, and suggest a novel pathway for regulating the activity of cargo-bound kinesin. Work supported by NIGMS grants GM64624 to SPG, GM74830-06A1 to LH, GM76516 to LB, NS048501 to SJK, and AHA grant 825278F to JX.

Molecular origin of the weak susceptibility of kinesin velocity to loads and its relation to the collective behavior of kinesins

PubMed Central

Wang, Qian; Diehl, Michael R.; Jana, Biman; Cheung, Margaret S.; Kolomeisky, Anatoly B.; Onuchic, José N.

2017-01-01

Motor proteins are active enzymatic molecules that support important cellular processes by transforming chemical energy into mechanical work. Although the structures and chemomechanical cycles of motor proteins have been extensively investigated, the sensitivity of a motor’s velocity in response to a force is not well-understood. For kinesin, velocity is weakly influenced by a small to midrange external force (weak susceptibility) but is steeply reduced by a large force. Here, we utilize a structure-based molecular dynamic simulation to study the molecular origin of the weak susceptibility for a single kinesin. We show that the key step in controlling the velocity of a single kinesin under an external force is the ATP release from the microtubule-bound head. Only under large loading forces can the motor head release ATP at a fast rate, which significantly reduces the velocity of kinesin. It underpins the weak susceptibility that the velocity will not change at small to midrange forces. The molecular origin of this velocity reduction is that the neck linker of a kinesin only detaches from the motor head when pulled by a large force. This prompts the ATP binding site to adopt an open state, favoring ATP release and reducing the velocity. Furthermore, we show that two load-bearing kinesins are incapable of equally sharing the load unless they are very close to each other. As a consequence of the weak susceptibility, the trailing kinesin faces the challenge of catching up to the leading one, which accounts for experimentally observed weak cooperativity of kinesins motors. PMID:28973894

Potential involvement of kinesin-1 in the regulation of subcellular localization of Girdin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muramatsu, Aya; Enomoto, Atsushi, E-mail: enomoto@iar.nagoya-u.ac.jp; Kato, Takuya

Girdin is an actin-binding protein that has multiple functions in postnatal neural development and cancer progression. We previously showed that Girdin is a regulator of migration for neuroblasts born from neural stem cells in the subventricular zone (SVZ) and the dentate gyrus of the hippocampus in the postnatal brain. Despite a growing list of Girdin-interacting proteins, the mechanism of Girdin-mediated migration has not been fully elucidated. Girdin interacts with Disrupted-In-Schizophrenia 1 and partitioning-defective 3, both of which have been shown to interact with the kinesin microtubule motor proteins. Based on this, we have identified that Girdin also interacts with kinesin-1,more » a member of neuronal kinesin proteins. Although a direct interaction of Girdin and kinesin-1 has not been determined, it is of interest to find that Girdin loss-of-function mutant mice with the mutation of a basic amino acid residue-rich region (Basic mut mice) exhibit limited interaction with kinesin-1. Furthermore, expression of a kinesin-1 mutant with motor defects, leads to Girdin mislocalization. Finally, consistent with previous studies on the role of kinesin proteins in trafficking a cell–cell adhesion molecule N-cadherin, Basic mut mice showed an aberrant expression pattern of N-cadherin in migrating SVZ neuroblasts. These findings suggest a potential role of Girdin/kinesin-1 interaction in the regulation of neuroblast migration in the postnatal brain. - Highlights: • Girdin is a regulator of migration for neuroblasts in the postnatal brain. • Girdin interacts with kinesin-1, a member of neuronal kinesin proteins. • Girdin mutant mice showed an aberrant expression of N-cadherin in neuroblasts.« less

Vaccinia virus proteins A36 and F12/E2 show strong preferences for different kinesin light chain isoforms.

PubMed

Gao, William N D; Carpentier, David C J; Ewles, Helen A; Lee, Stacey-Ann; Smith, Geoffrey L

2017-08-01

Vaccinia virus (VACV) utilizes microtubule-mediated trafficking at several stages of its life cycle, of which virus egress is the most intensely studied. During egress VACV proteins A36, F12 and E2 are involved in kinesin-1 interactions; however, the roles of these proteins remain poorly understood. A36 forms a direct link between virions and kinesin-1, yet in its absence VACV egress still occurs on microtubules. During a co-immunoprecipitation screen to seek an alternative link between virions and kinesin, A36 was found to bind isoform KLC1 rather than KLC2. The F12/E2 complex associates preferentially with the C-terminal tail of KLC2, to a region that overlaps the binding site of cellular 14-3-3 proteins. F12/E2 displaces 14-3-3 from KLC and, unlike 14-3-3, does not require phosphorylation of KLC for its binding. The region determining the KLC1 specificity of A36 was mapped to the KLC N-terminal heptad repeat region that is responsible for its association with kinesin heavy chain. Despite these differing binding properties F12/E2 can co-operatively enhance A36 association with KLC, particularly when using a KLC1-KLC2 chimaera that resembles several KLC1 spliceforms and can bind A36 and F12/E2 efficiently. This is the first example of a pathogen encoding multiple proteins that co-operatively associate with kinesin-1. © 2017 The Authors. Traffic published by John Wiley & Sons Ltd.

Kinesin Steps Do Not Alternate in Size☆

PubMed Central

Fehr, Adrian N.; Asbury, Charles L.; Block, Steven M.

2008-01-01

Abstract Kinesin is a two-headed motor protein that transports cargo inside cells by moving stepwise on microtubules. Its exact trajectory along the microtubule is unknown: alternative pathway models predict either uniform 8-nm steps or alternating 7- and 9-nm steps. By analyzing single-molecule stepping traces from “limping” kinesin molecules, we were able to distinguish alternate fast- and slow-phase steps and thereby to calculate the step sizes associated with the motions of each of the two heads. We also compiled step distances from nonlimping kinesin molecules and compared these distributions against models predicting uniform or alternating step sizes. In both cases, we find that kinesin takes uniform 8-nm steps, a result that strongly constrains the allowed models. PMID:18083906

A Kinesin-Related Protein Required for the Mitotic Spindle Assembly

DTIC Science & Technology

1999-05-01

8217 at 23 °C. The residual 20 ul can be frozen in liquid nitrogen and used to estimate total recovery if desired. 3. Remove supernatant as thoroughly...communicated to the XKCM1 protein, and what can these results tell us about how chromosomes are segregated? These are questions that are currently...METHODS 3.1 Preparation of Microtubule Substrates The effects of kinesins on MT dynamics can be assayed by using three different types of MT

Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast

PubMed Central

Gergely, Zachary R.; Crapo, Ammon; Hough, Loren E.; McIntosh, J. Richard; Betterton, Meredith D.

2016-01-01

Kinesin-8 motor proteins destabilize microtubules. Their absence during cell division is associated with disorganized mitotic chromosome movements and chromosome loss. Despite recent work studying effects of kinesin-8s on microtubule dynamics, it remains unclear whether the kinesin-8 mitotic phenotypes are consequences of their effect on microtubule dynamics, their well-established motor activity, or additional, unknown functions. To better understand the role of kinesin-8 proteins in mitosis, we studied the effects of deletion of the fission yeast kinesin-8 proteins Klp5 and Klp6 on chromosome movements and spindle length dynamics. Aberrant microtubule-driven kinetochore pushing movements and tripolar mitotic spindles occurred in cells lacking Klp5 but not Klp6. Kinesin-8–deletion strains showed large fluctuations in metaphase spindle length, suggesting a disruption of spindle length stabilization. Comparison of our results from light microscopy with a mathematical model suggests that kinesin-8–induced effects on microtubule dynamics, kinetochore attachment stability, and sliding force in the spindle can explain the aberrant chromosome movements and spindle length fluctuations seen. PMID:27146110

Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins.

PubMed

Saritas-Yildirim, Banu; Pliner, Hannah A; Ochoa, Angelica; Silva, Elena M

2015-01-01

Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.

Kinesin-1 and mitochondrial motility control by discrimination of structurally equivalent but distinct subdomains in Ran-GTP-binding domains of Ran-binding protein 2.

PubMed

Patil, Hemangi; Cho, Kyoung-in; Lee, James; Yang, Yi; Orry, Andrew; Ferreira, Paulo A

2013-03-27

The pleckstrin homology (PH) domain is a versatile fold that mediates a variety of protein-protein and protein-phosphatidylinositol lipid interactions. The Ran-binding protein 2 (RanBP2) contains four interspersed Ran GTPase-binding domains (RBD(n = 1-4)) with close structural homology to the PH domain of Bruton's tyrosine kinase. The RBD2, kinesin-binding domain (KBD) and RBD3 comprise a tripartite domain (R2KR3) of RanBP2 that causes the unfolding, microtubule binding and biphasic activation of kinesin-1, a crucial anterograde motor of mitochondrial motility. However, the interplay between Ran GTPase and R2KR3 of RanBP2 in kinesin-1 activation and mitochondrial motility is elusive. We use structure-function, biochemical, kinetic and cell-based assays with time-lapse live-cell microscopy of over 260,000 mitochondrial-motility-related events to find mutually exclusive subdomains in RBD2 and RBD3 towards Ran GTPase binding, kinesin-1 activation and mitochondrial motility regulation. The RBD2 and RBD3 exhibit Ran-GTP-independent, subdomain and stereochemical-dependent discrimination on the biphasic kinetics of kinesin-1 activation or regulation of mitochondrial motility. Further, KBD alone and R2KR3 stimulate and suppress, respectively, multiple biophysical parameters of mitochondrial motility. The regulation of the bidirectional transport of mitochondria by either KBD or R2KR3 is highly coordinated, because their kinetic effects are accompanied always by changes in mitochondrial motile events of either transport polarity. These studies uncover novel roles in Ran GTPase-independent subdomains of RBD2 and RBD3, and KBD of RanBP2, that confer antagonizing and multi-modal mechanisms of kinesin-1 activation and regulation of mitochondrial motility. These findings open new venues towards the pharmacological harnessing of cooperative and competitive mechanisms regulating kinesins, RanBP2 or mitochondrial motility in disparate human disorders.

Chlorpyrifos, chlorpyrifos-oxon, and diisopropylfluorophosphate inhibit kinesin-dependent microtubule motility

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gearhart, Debra A.; Sickles, Dale W.; Buccafusco, Jerry J.

2007-01-01

Diisopropylfluorophosphate, originally developed as a chemical warfare agent, is structurally similar to nerve agents, and chlorpyrifos has extensive worldwide use as an agricultural pesticide. While inhibition of cholinesterases underlies the acute toxicity of these organophosphates, we previously reported impaired axonal transport in the sciatic nerves from rats treated chronically with subthreshold doses of chlorpyrifos. Those data indicate that chlorpyrifos (and/or its active metabolite, chlorpyrifos-oxon) might directly affect the function of kinesin and/or microtubules-the principal proteins that mediate anterograde axonal transport. The current report describes in vitro assays to assess the concentration-dependent effects of chlorpyrifos (0-10 {mu}M), chlorpyrifos-oxon (0-10 {mu}M), andmore » diisopropylfluorophosphate (0-0.59 nM) on kinesin-dependent microtubule motility. Preincubating bovine brain microtubules with the organophosphates did not alter kinesin-mediated microtubule motility. In contrast, preincubation of bovine brain kinesin with diisopropylfluorophosphate, chlorpyrifos, or chlorpyrifos-oxon produced a concentration-dependent increase in the number of locomoting microtubules that detached from the kinesin-coated glass cover slip. Our data suggest that the organophosphates-chlorpyrifos-oxon, chlorpyrifos, and diisopropylfluorophosphate-directly affect kinesin, thereby disrupting kinesin-dependent transport on microtubules. Kinesin-dependent movement of vesicles, organelles, and other cellular components along microtubules is fundamental to the organization of all eukaryotic cells, especially in neurons where organelles and proteins synthesized in the cell body must move down long axons to pre-synaptic sites in nerve terminals. We postulate that disruption of kinesin-dependent intracellular transport could account for some of the long-term effects of organophosphates on the peripheral and central nervous system.« less

Structural analysis of intermolecular interactions in the kinesin adaptor complex fasciculation and elongation protein zeta 1/ short coiled-coil protein (FEZ1/SCOCO).

PubMed

Alborghetti, Marcos Rodrigo; Furlan, Ariane da Silva; da Silva, Júlio César; Sforça, Maurício Luís; Honorato, Rodrigo Vargas; Granato, Daniela Campos; dos Santos Migueleti, Deivid Lucas; Neves, Jorge L; de Oliveira, Paulo Sergio Lopes; Paes-Leme, Adriana Franco; Zeri, Ana Carolina de Mattos; de Torriani, Iris Concepcion Linares; Kobarg, Jörg

2013-01-01

Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.

Structural Analysis of Intermolecular Interactions in the Kinesin Adaptor Complex Fasciculation and Elongation Protein Zeta 1/ Short Coiled-Coil Protein (FEZ1/SCOCO)

PubMed Central

da Silva, Júlio César; Sforça, Maurício Luís; Honorato, Rodrigo Vargas; Granato, Daniela Campos; dos Santos Migueleti, Deivid Lucas; Neves, Jorge L.; de Oliveira, Paulo Sergio Lopes; Paes-Leme, Adriana Franco; Zeri, Ana Carolina de Mattos; de Torriani, Iris Concepcion Linares; Kobarg, Jörg

2013-01-01

Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth. PMID:24116125

A kinesin-1 binding motif in vaccinia virus that is widespread throughout the human genome

PubMed Central

Dodding, Mark P; Mitter, Richard; Humphries, Ashley C; Way, Michael

2011-01-01

Transport of cargoes by kinesin-1 is essential for many cellular processes. Nevertheless, the number of proteins known to recruit kinesin-1 via its cargo binding light chain (KLC) is still quite small. We also know relatively little about the molecular features that define kinesin-1 binding. We now show that a bipartite tryptophan-based kinesin-1 binding motif, originally identified in Calsyntenin is present in A36, a vaccinia integral membrane protein. This bipartite motif in A36 is required for kinesin-1-dependent transport of the virus to the cell periphery. Bioinformatic analysis reveals that related bipartite tryptophan-based motifs are present in over 450 human proteins. Using vaccinia as a surrogate cargo, we show that regions of proteins containing this motif can function to recruit KLC and promote virus transport in the absence of A36. These proteins interact with the kinesin light chain outside the context of infection and have distinct preferences for KLC1 and KLC2. Our observations demonstrate that KLC binding can be conferred by a common set of features that are found in a wide range of proteins associated with diverse cellular functions and human diseases. PMID:21915095

Conservation and divergence of ADAM family proteins in the Xenopus genome

PubMed Central

2010-01-01

Background Members of the disintegrin metalloproteinase (ADAM) family play important roles in cellular and developmental processes through their functions as proteases and/or binding partners for other proteins. The amphibian Xenopus has long been used as a model for early vertebrate development, but genome-wide analyses for large gene families were not possible until the recent completion of the X. tropicalis genome sequence and the availability of large scale expression sequence tag (EST) databases. In this study we carried out a systematic analysis of the X. tropicalis genome and uncovered several interesting features of ADAM genes in this species. Results Based on the X. tropicalis genome sequence and EST databases, we identified Xenopus orthologues of mammalian ADAMs and obtained full-length cDNA clones for these genes. The deduced protein sequences, synteny and exon-intron boundaries are conserved between most human and X. tropicalis orthologues. The alternative splicing patterns of certain Xenopus ADAM genes, such as adams 22 and 28, are similar to those of their mammalian orthologues. However, we were unable to identify an orthologue for ADAM7 or 8. The Xenopus orthologue of ADAM15, an active metalloproteinase in mammals, does not contain the conserved zinc-binding motif and is hence considered proteolytically inactive. We also found evidence for gain of ADAM genes in Xenopus as compared to other species. There is a homologue of ADAM10 in Xenopus that is missing in most mammals. Furthermore, a single scaffold of X. tropicalis genome contains four genes encoding ADAM28 homologues, suggesting genome duplication in this region. Conclusions Our genome-wide analysis of ADAM genes in X. tropicalis revealed both conservation and evolutionary divergence of these genes in this amphibian species. On the one hand, all ADAMs implicated in normal development and health in other species are conserved in X. tropicalis. On the other hand, some ADAM genes and ADAM protease

Neck linker length determines the degree of processivity in kinesin-1 and kinesin-2 motors.

PubMed

Shastry, Shankar; Hancock, William O

2010-05-25

Defining the mechanical and biochemical determinates of kinesin processivity is important for understanding how diverse kinesins are tuned for specific cellular functions. Because transmission of mechanical forces through the 14-18 amino acid neck linker domain underlies coordinated stepping, we investigated the role of neck linker length, charge, and structure in kinesin-1 and kinesin-2 motor behavior. For optimum comparison with kinesin-1, the KIF3A head and neck linker of kinesin-2 were fused to the kinesin-1 neck coil and rod. Extending the 14-residue kinesin-1 neck linker reduced processivity, and shortening the 17-residue kinesin-2 neck linker enhanced processivity. When a proline in the kinesin-2 neck linker was replaced, kinesin-1 and kinesin-2 run lengths scaled identically with neck linker length, despite moving at different speeds. In low-ionic-strength buffer, charge had a dominant effect on motor processivity, which resolves ongoing controversy regarding the effect of neck linker length on kinesin processivity. From stochastic simulations, the results are best explained by neck linker extension slowing strain-dependent detachment of the rear head along with diminishing strain-dependent inhibition of ATP binding. These results help delineate how interhead strain maximizes stepping and suggest that less processive kinesins are tuned to coordinate with other motors differently than the maximally processive kinesin-1. Copyright 2010 Elsevier Ltd. All rights reserved.

Hook is an adapter that coordinates kinesin-3 and dynein cargo attachment on early endosomes

PubMed Central

Bielska, Ewa; Schuster, Martin; Roger, Yvonne; Berepiki, Adokiye; Soanes, Darren M.; Talbot, Nicholas J.

2014-01-01

Bidirectional membrane trafficking along microtubules is mediated by kinesin-1, kinesin-3, and dynein. Several organelle-bound adapters for kinesin-1 and dynein have been reported that orchestrate their opposing activity. However, the coordination of kinesin-3/dynein-mediated transport is not understood. In this paper, we report that a Hook protein, Hok1, is essential for kinesin-3– and dynein-dependent early endosome (EE) motility in the fungus Ustilago maydis. Hok1 binds to EEs via its C-terminal region, where it forms a complex with homologues of human fused toes (FTS) and its interactor FTS- and Hook-interacting protein. A highly conserved N-terminal region is required to bind dynein and kinesin-3 to EEs. To change the direction of EE transport, kinesin-3 is released from organelles, and dynein binds subsequently. A chimaera of human Hook3 and Hok1 rescues the hok1 mutant phenotype, suggesting functional conservation between humans and fungi. We conclude that Hok1 is part of an evolutionarily conserved protein complex that regulates bidirectional EE trafficking by controlling attachment of both kinesin-3 and dynein. PMID:24637326

Processive movement of single kinesins on crowded microtubules visualized using quantum dots

PubMed Central

Seitz, Arne; Surrey, Thomas

2006-01-01

Kinesin-1 is a processive molecular motor transporting cargo along microtubules. Inside cells, several motors and microtubule-associated proteins compete for binding to microtubules. Therefore, the question arises how processive movement of kinesin-1 is affected by crowding on the microtubule. Here we use total internal reflection fluorescence microscopy to image in vitro the runs of single quantum dot-labelled kinesins on crowded microtubules under steady-state conditions and to measure the degree of crowding on a microtubule at steady-state. We find that the runs of kinesins are little affected by high kinesin densities on a microtubule. However, the presence of high densities of a mutant kinesin that is not able to step efficiently reduces the average speed of wild-type kinesin, while hardly changing its processivity. This indicates that kinesin waits in a strongly bound state on the microtubule when encountering an obstacle until the obstacle unbinds and frees the binding site for kinesin's next step. A simple kinetic model can explain quantitatively the behaviour of kinesin under both crowding conditions. PMID:16407972

Analysis of molecular chaperones using a Xenopus oocyte protein refolding assay.

PubMed

Heikkila, John J; Kaldis, Angelo; Abdulle, Rashid

2006-01-01

Heat shock proteins (Hsps) are molecular chaperones that aid in the folding and translocation of protein under normal conditions and protect cellular proteins during stressful situations. A family of Hsps, the small Hsps, can maintain denatured target proteins in a folding-competent state such that they can be refolded and regain biological activity in the presence of other molecular chaperones. Previous assays have employed cellular lysates as a source of molecular chaperones involved in folding. In this chapter, we describe the production and purification of a Xenopus laevis recombinant small Hsp, Hsp30C, and an in vivo luciferase (LUC) refolding assay employing microinjected Xenopus oocytes. This assay tests whether LUC can be maintained in a folding-competent state when heat denatured in the presence of a small Hsp or other molecular chaperone. For example, micro-injection of heat-denatured LUC alone into oocytes resulted in minimal reactivation of enzyme activity. However, LUC heat denatured in the presence of Hsp30C resulted in 100% recovery of enzyme activity after microinjection. The in vivo oocyte refolding system is more sensitive and requires less molecular chaperone than in vitro refolding assays. Also, this protocol is not limited to testing Xenopus molecular chaperones because small Hsps from other organisms have been used successfully.

NtKRP, a kinesin-12 protein, regulates embryo/seed size and seed germination via involving in cell cycle progression at the G2/M transition.

PubMed

Tian, Shujuan; Wu, Jingjing; Li, Fen; Zou, Jianwei; Liu, Yuwen; Zhou, Bing; Bai, Yang; Sun, Meng-Xiang

2016-10-25

Kinesins comprise a superfamily of microtubule-based motor proteins involved in essential processes in plant development, but few kinesins have been functionally identified during seed development. Especially, few kinesins that regulate cell division during embryogenesis have been identified. Here we report the functional characterization of NtKRP, a motor protein of the kinesin-12 family. NtKRP is predominantly expressed in embryos and embryonic roots. NtKRP RNAi lines displayed reductions in cell numbers in the meristematic zone, in embryonic root length, and in mature embryo and seed sizes. Furthermore, we also show that CDKA;1 binds to NtKRP at the consensus phosphorylation sites and that the decreased cell numbers in NtKRP-silenced embryos are due to a delay in cell division cycle at the G2/M transition. In addition, binding between the cargo-binding tail domain of NtKRP and CDKA; 1 was also determined. Our results reveal a novel molecular pathway that regulates embryo/seed development and critical role of kinesin in temporal and spatial regulation of a specific issue of embryo developmental.

Two Heteromeric Kinesin Complexes in Chemosensory Neurons and Sensory Cilia of Caenorhabditis elegans

PubMed Central

Signor, Dawn; Wedaman, Karen P.; Rose, Lesilee S.; Scholey, Jonathan M.

1999-01-01

Chemosensation in the nervous system of the nematode Caenorhabditis elegans depends on sensory cilia, whose assembly and maintenance requires the transport of components such as axonemal proteins and signal transduction machinery to their site of incorporation into ciliary structures. Members of the heteromeric kinesin family of microtubule motors are prime candidates for playing key roles in these transport events. Here we describe the molecular characterization and partial purification of two heteromeric kinesin complexes from C. elegans, heterotrimeric CeKinesin-II and dimeric CeOsm-3. Transgenic worms expressing green fluorescent protein driven by endogenous heteromeric kinesin promoters reveal that both CeKinesin-II and CeOsm-3 are expressed in amphid, inner labial, and phasmid chemosensory neurons. Additionally, immunolocalization experiments on fixed worms show an intense concentration of CeKinesin-II and CeOsm-3 polypeptides in the ciliated endings of these chemosensory neurons and a punctate localization pattern in the corresponding cell bodies and dendrites. These results, together with the phenotypes of known mutants in the pathway of sensory ciliary assembly, suggest that CeKinesin-II and CeOsm-3 drive the transport of ciliary components required for sequential steps in the assembly of chemosensory cilia. PMID:9950681

Casein Kinase 2 Reverses Tail-Independent Inhibition of Kinesin-1

NASA Astrophysics Data System (ADS)

Xu, Jing; Shu, Zhanyong; Anand, Preetha; Reddy, Babu; Cermelli, Silvia; Whisenant, Thomas; King, Stephen; Bardwell, Lee; Huang, Lan; Gross, Steven

2011-03-01

Kinesin-1 is a plus-end microtubule-based molecular motor, and defects in kinesin transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a direct head-tail interaction, but is believed to be active otherwise. In contrast, this study uncovers a fast but reversible inhibition distinct from the canonical auto-inhibition pathway. The majority of the initially active kinesin (full-length or tail-less) loses its ability to bind/interact with microtubule, and Casein Kinase 2 (CK2) reverses this inactivation (up to 4-fold) without altering kinesin's single motor properties. Motor phosphorylation is not required for this CK2 -mediated kinesin activation. In cultured mammalian cells, knockdown of CK2 level, but not kinase activity, was sufficient to decrease the force required to stall lipid droplet transport, consistent with a reduction in the number of active motors. We propose that CK2 forms a positive regulating complex with the motor. This study provides the first direct evidence of a protein kinase positively regulating kinesin-transport, and uncovers a pathway whereby inactive cargo-bound kinesin can be activated. This work is supported by NIGMS grants GM64624 and GM079156 to SPG, GM-74830 to LH, NIH grants GM76516 and GM60366 to LB, and AHA grant 825278F to JX.

The KAC family of kinesin-like proteins is essential for the association of chloroplasts with the plasma membrane in land plants.

PubMed

Suetsugu, Noriyuki; Sato, Yoshikatsu; Tsuboi, Hidenori; Kasahara, Masahiro; Imaizumi, Takato; Kagawa, Takatoshi; Hiwatashi, Yuji; Hasebe, Mitsuyasu; Wada, Masamitsu

2012-11-01

Chloroplasts require association with the plasma membrane for movement in response to light and for appropriate positioning within the cell to capture photosynthetic light efficiently. In Arabidopsis, CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for both the proper movement of chloroplasts and the association of chloroplasts with the plasma membrane, through the reorganization of short actin filaments located on the periphery of the chloroplasts. Here, we show that KAC and CHUP1 orthologs (AcKAC1, AcCHUP1A and AcCHUP1B, and PpKAC1 and PpKAC2) play important roles in chloroplast positioning in the fern Adiantum capillus-veneris and the moss Physcomitrella patens. The knockdown of AcKAC1 and two AcCHUP1 genes induced the aggregation of chloroplasts around the nucleus. Analyses of A. capillus-veneris mutants containing perinuclear-aggregated chloroplasts confirmed that AcKAC1 is required for chloroplast-plasma membrane association. In addition, P. patens lines in which two KAC genes had been knocked out showed an aggregated chloroplast phenotype similar to that of the fern kac1 mutants. These results indicate that chloroplast positioning and movement are mediated through the activities of KAC and CHUP1 proteins, which are conserved in land plants.

Coupling of kinesin ATP turnover to translocation and microtubule regulation: one engine, many machines.

PubMed

Friel, Claire T; Howard, Jonathon

2012-12-01

The cycle of ATP turnover is integral to the action of motor proteins. Here we discuss how variation in this cycle leads to variation of function observed amongst members of the kinesin superfamily of microtubule associated motor proteins. Variation in the ATP turnover cycle among superfamily members can tune the characteristic kinesin motor to one of the range of microtubule-based functions performed by kinesins. The speed at which ATP is hydrolysed affects the speed of translocation. The ratio of rate constants of ATP turnover in relation to association and dissociation from the microtubule influence the processivity of translocation. Variation in the rate-limiting step of the cycle can reverse the way in which the motor domain interacts with the microtubule producing non-motile kinesins. Because the ATP turnover cycle is not fully understood for the majority of kinesins, much work remains to show how the kinesin engine functions in such a wide variety of molecular machines.

Intracellular Transport and Kinesin Superfamily Proteins: Structure, Function and Dynamics

NASA Astrophysics Data System (ADS)

Hirokawa, N.; Takemura, R.

Using various molecular cell biological and molecular genetic approaches, we identified kinesin superfamily proteins (KIFs) and characterized their significant functions in intracellular transport, which is fundamental for cellular morphogenesis, functioning, and survival. We showed that KIFs not only transport various membranous organelles, proteins complexes and mRNAs fundamental for cellular functions but also play significant roles in higher brain functions such as memory and learning, determination of important developmental processes such as left-right asymmetry formation and brain wiring. We also elucidated that KIFs recognize and bind to their specific cargoes using scaffolding or adaptor protein complexes. Concerning the mechanism of motility, we discovered the simplest unique monomeric motor KIF1A and determined by molecular biophysics, cryoelectron microscopy and X-ray crystallography that KIF1A can move on a microtubule processively as a monomer by biased Brownian motion and by hydolyzing ATP.

Maternal syntabulin is required for dorsal axis formation and is a germ plasm component in Xenopus.

PubMed

Colozza, Gabriele; De Robertis, Edward M

2014-07-01

In amphibians and teleosts, early embryonic axial development is driven by maternally deposited mRNAs and proteins, called dorsal determinants, which migrate to the presumptive dorsal side of the embryo in a microtubule-dependent manner after fertilization. Syntabulin is an adapter protein that binds to kinesin KIF5B and to the transmembrane protein Syntaxin1. In zebrafish, a mutation in Syntabulin causes complete embryo ventralization. It is unknown whether Syntabulin plays an analogous role during early development of other species, a question addressed here in Xenopus laevis. in situ hybridization of syntabulin mRNA was carried out at different stages of Xenopus development. In oocytes, syntabulin transcripts were localized to the vegetal cortex of large oocytes and the mitochondrial cloud of very young oocytes. We extended the zebrafish data by finding that during cleavage Xenopus syntabulin mRNA localized to the germ plasm and was later expressed in primordial germ cells (PGCs). This new finding suggested a role for Syntabulin during germ cell differentiation. The functional role of maternal syntabulin mRNA was investigated by knock-down with phosphorothioate DNA antisense oligos followed by oocyte transfer. The results showed that syntabulin mRNA depletion caused the complete loss of dorso-anterior axis formation in frog embryos. Consistent with the ventralized phenotype, syntabulin-depleted embryos displayed severe reduction of dorsal markers and ubiquitous transcription of the ventral marker sizzled. Syntabulin was required for the maternal Wnt/β-Catenin signal, since ventralization could be completely rescued by injection of β-catenin (or syntabulin) mRNA. The data suggest an evolutionarily conserved role for Syntabulin, a protein that bridges microtubule motors and membrane vesicles, during dorso-ventral axis formation in the vertebrates. Copyright © 2013 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

The beginning of kinesin's force-generating cycle visualized at 9-Å resolution

PubMed Central

Sindelar, Charles V.; Downing, Kenneth H.

2007-01-01

We have used cryo-electron microscopy of kinesin-decorated microtubules to resolve the structure of the motor protein kinesin's crucial nucleotide response elements, switch I and the switch II helix, in kinesin's poorly understood nucleotide-free state. Both of the switch elements undergo conformational change relative to the microtubule-free state. The changes in switch I suggest a role for it in “ejecting” adenosine diphosphate when kinesin initially binds to the microtubule. The switch II helix has an N-terminal extension, apparently stabilized by conserved microtubule contacts, implying a microtubule activation mechanism that could convey the state of the bound nucleotide to kinesin's putative force-delivering element (the “neck linker”). In deriving this structure, we have adapted an image-processing technique, single-particle reconstruction, for analyzing decorated microtubules. The resulting reconstruction visualizes the asymmetric seam present in native, 13-protofilament microtubules, and this method will provide an avenue to higher-resolution characterization of a variety of microtubule- binding proteins, as well as the microtubule itself. PMID:17470637

Orphan Kinesin NOD Lacks Motile Properties But Does Possess a Microtubule-stimulated ATPase Activity

PubMed Central

Matthies, Heinrich J.G.; Baskin, Ronald J.; Hawley, R. Scott

2001-01-01

NOD is a Drosophila chromosome-associated kinesin-like protein that does not fall into the chromokinesin subfamily. Although NOD lacks residues known to be critical for kinesin function, we show that microtubules activate the ATPase activity of NOD >2000-fold. Biochemical and genetic analysis of two genetically identified mutations of NOD (NODDTW and NOD“DR2”) demonstrates that this allosteric activation is critical for the function of NOD in vivo. However, several lines of evidence indicate that this ATPase activity is not coupled to vectorial transport, including 1) NOD does not produce microtubule gliding; and 2) the substitution of a single amino acid in the Drosophila kinesin heavy chain with the analogous amino acid in NOD results in a drastic inhibition of motility. We suggest that the microtubule-activated ATPase activity of NOD provides transient attachments of chromosomes to microtubules rather than producing vectorial transport. PMID:11739796

Small Molecule Screen for Candidate Antimalarials Targeting Plasmodium Kinesin-5*

PubMed Central

Liu, Liqiong; Richard, Jessica; Kim, Sunyoung; Wojcik, Edward J.

2014-01-01

Plasmodium falciparum and vivax are responsible for the majority of malaria infections worldwide, resulting in over a million deaths annually. Malaria parasites now show measured resistance to all currently utilized drugs. Novel antimalarial drugs are urgently needed. The Plasmodium Kinesin-5 mechanoenzyme is a suitable “next generation” target. Discovered via small molecule screen experiments, the human Kinesin-5 has multiple allosteric sites that are “druggable.” One site in particular, unique in its sequence divergence across all homologs in the superfamily and even within the same family, exhibits exquisite drug specificity. We propose that Plasmodium Kinesin-5 shares this allosteric site and likewise can be targeted to uncover inhibitors with high specificity. To test this idea, we performed a screen for inhibitors selective for Plasmodium Kinesin-5 ATPase activity in parallel with human Kinesin-5. Our screen of nearly 2000 compounds successfully identified compounds that selectively inhibit both P. vivax and falciparum Kinesin-5 motor domains but, as anticipated, do not impact human Kinesin-5 activity. Of note is a candidate drug that did not biochemically compete with the ATP substrate for the conserved active site or disrupt the microtubule-binding site. Together, our experiments identified MMV666693 as a selective allosteric inhibitor of Plasmodium Kinesin-5; this is the first identified protein target for the Medicines of Malaria Venture validated collection of parasite proliferation inhibitors. This work demonstrates that chemical screens against human kinesins are adaptable to homologs in disease organisms and, as such, extendable to strategies to combat infectious disease. PMID:24737313

Surface-Bound Casein Modulates the Adsorption and Activity of Kinesin on SiO2 Surfaces

PubMed Central

Ozeki, Tomomitsu; Verma, Vivek; Uppalapati, Maruti; Suzuki, Yukiko; Nakamura, Mikihiko; Catchmark, Jeffrey M.; Hancock, William O.

2009-01-01

Abstract Conventional kinesin is routinely adsorbed to hydrophilic surfaces such as SiO2. Pretreatment of surfaces with casein has become the standard protocol for achieving optimal kinesin activity, but the mechanism by which casein enhances kinesin surface adsorption and function is poorly understood. We used quartz crystal microbalance measurements and microtubule gliding assays to uncover the role that casein plays in enhancing the activity of surface-adsorbed kinesin. On SiO2 surfaces, casein adsorbs as both a tightly bound monolayer and a reversibly bound second layer that has a dissociation constant of 500 nM and can be desorbed by washing with casein-free buffer. Experiments using truncated kinesins demonstrate that in the presence of soluble casein, kinesin tails bind well to the surface, whereas kinesin head binding is blocked. Removing soluble casein reverses these binding profiles. Surprisingly, reversibly bound casein plays only a moderate role during kinesin adsorption, but it significantly enhances kinesin activity when surface-adsorbed motors are interacting with microtubules. These results point to a model in which a dynamic casein bilayer prevents reversible association of the heads with the surface and enhances association of the kinesin tail with the surface. Understanding protein-surface interactions in this model system should provide a framework for engineering surfaces for functional adsorption of other motor proteins and surface-active enzymes. PMID:19383474

Distribution of tubulin, kinesin, and dynein in light- and dark-adapted octopus retinas.

PubMed

Martinez, J M; Elfarissi, H; De Velasco, B; Ochoa, G H; Miller, A M; Clark, Y M; Matsumoto, B; Robles, L J

2000-01-01

Cephalopod retinas exhibit several responses to light and dark adaptation, including rhabdom size changes, photopigment movements, and pigment granule migration. Light- and dark-directed rearrangements of microfilament and microtubule cytoskeletal transport pathways could drive these changes. Recently, we localized actin-binding proteins in light-/dark-adapted octopus rhabdoms and suggested that actin cytoskeletal rearrangements bring about the formation and degradation of rhabdomere microvilli subsets. To determine if the microtubule cytoskeleton and associated motor proteins control the other light/dark changes, we used immunoblotting and immunocytochemical procedures to map the distribution of tubulin, kinesin, and dynein in dorsal and ventral halves of light- and dark-adapted octopus retinas. Immunoblots detected alpha- and beta-tubulin, dynein intermediate chain, and kinesin heavy chain in extracts of whole retinas. Epifluorescence and confocal microscopy showed that the tubulin proteins were distributed throughout the retina with more immunoreactivity in retinas exposed to light. Kinesin localization was heavy in the pigment layer of light- and dark-adapted ventral retinas but was less prominent in the dorsal region. Dynein distribution also varied in dorsal and ventral retinas with more immunoreactivity in light- and dark-adapted ventral retinas and confocal microscopy emphasized the granular nature of this labeling. We suggest that light may regulate the distribution of microtubule cytoskeletal proteins in the octopus retina and that position, dorsal versus ventral, also influences the distribution of motor proteins. The microtubule cytoskeleton is most likely involved in pigment granule migration in the light and dark and with the movement of transport vesicles from the photoreceptor inner segments to the rhabdoms.

Identification of globular mechanochemical heads of kinesin.

PubMed

Scholey, J M; Heuser, J; Yang, J T; Goldstein, L S

1989-03-23

Kinesin is a mechanoenzyme which uses energy liberated from ATP hydrolysis to transport particles towards the 'plus ends' of microtubules. The enzyme consists of two polypeptide heavy chains of relative molecular mass (Mr) approximately 110,000-140,000 (110K-140K) plus copurifying light chains; these polypeptides are arranged in a structure consisting of two globular heads attached to a fibrous stalk which terminates in a 'feathered' tail. Here we report that a function-disrupting monoclonal antikinesin, which binds to the 45K fragment of the kinesin heavy chain, recognizes an epitope located towards the N-terminal end of the heavy chain, and decorates the two globular heads lying at one end of the intact molecules (one antibody per head). The results show that the two heavy chains of native kinesin are arranged in parallel, and that the 45K fragments, which display nucleotide-sensitive interactions with microtubules, represent mechanochemical 'heads' located at the N-terminal regions of the heavy chains. Thus, it is likely that the kinesin heads are analogous to the subfragment-1 domains of myosin.

Cargo selection by specific kinesin light chain 1 isoforms.

PubMed

Woźniak, Marcin J; Allan, Victoria J

2006-11-29

Kinesin-1 drives the movement of diverse cargoes, and it has been proposed that specific kinesin light chain (KLC) isoforms target kinesin-1 to these different structures. Here, we test this hypothesis using two in vitro motility assays, which reconstitute the movement of rough endoplasmic reticulum (RER) and vesicles present in a Golgi membrane fraction. We generated GST-tagged fusion proteins of KLC1B and KLC1D that included the tetratricopeptide repeat domain and the variable C-terminus. We find that preincubation of RER with KLC1B inhibits RER motility, whereas KLC1D does not. In contrast, Golgi fraction vesicle movement is inhibited by KLC1D but not KLC1B reagents. Both RER and vesicle movement is inhibited by preincubation with the GST-tagged C-terminal domain of ubiquitous kinesin heavy chain (uKHC), which binds to the N-terminal domain of uKHC and alters its interaction with microtubules. We propose that although the TRR domains are required for cargo binding, it is the variable C-terminal region of KLCs that are vital for targeting kinesin-1 to different cellular structures.

Cargo selection by specific kinesin light chain 1 isoforms

PubMed Central

Woźniak, Marcin J; Allan, Victoria J

2006-01-01

Kinesin-1 drives the movement of diverse cargoes, and it has been proposed that specific kinesin light chain (KLC) isoforms target kinesin-1 to these different structures. Here, we test this hypothesis using two in vitro motility assays, which reconstitute the movement of rough endoplasmic reticulum (RER) and vesicles present in a Golgi membrane fraction. We generated GST-tagged fusion proteins of KLC1B and KLC1D that included the tetratricopeptide repeat domain and the variable C-terminus. We find that preincubation of RER with KLC1B inhibits RER motility, whereas KLC1D does not. In contrast, Golgi fraction vesicle movement is inhibited by KLC1D but not KLC1B reagents. Both RER and vesicle movement is inhibited by preincubation with the GST-tagged C-terminal domain of ubiquitous kinesin heavy chain (uKHC), which binds to the N-terminal domain of uKHC and alters its interaction with microtubules. We propose that although the TRR domains are required for cargo binding, it is the variable C-terminal region of KLCs that are vital for targeting kinesin-1 to different cellular structures. PMID:17093494

Kinesin-dependent mechanism for controlling triglyceride secretion from the liver.

PubMed

Rai, Priyanka; Kumar, Mukesh; Sharma, Geetika; Barak, Pradeep; Das, Saumitra; Kamat, Siddhesh S; Mallik, Roop

2017-12-05

Despite massive fluctuations in its internal triglyceride content, the liver secretes triglyceride under tight homeostatic control. This buffering function is most visible after fasting, when liver triglyceride increases manyfold but circulating serum triglyceride barely fluctuates. How the liver controls triglyceride secretion is unknown, but is fundamentally important for lipid and energy homeostasis in animals. Here we find an unexpected cellular and molecular mechanism behind such control. We show that kinesin motors are recruited to triglyceride-rich lipid droplets (LDs) in the liver by the GTPase ARF1, which is a key activator of lipolysis. This recruitment is activated by an insulin-dependent pathway and therefore responds to fed/fasted states of the animal. In fed state, ARF1 and kinesin appear on LDs, consequently transporting LDs to the periphery of hepatocytes where the smooth endoplasmic reticulum (sER) is present. Because the lipases that catabolize LDs in hepatocytes reside on the sER, LDs can now be catabolized efficiently to provide triglyceride for lipoprotein assembly and secretion from the sER. Upon fasting, insulin is lowered to remove ARF1 and kinesin from LDs, thus down-regulating LD transport and sER-LD contacts. This tempers triglyceride availabiity for very low density lipoprotein assembly and allows homeostatic control of serum triglyceride in a fasted state. We further show that kinesin knockdown inhibits hepatitis-C virus replication in hepatocytes, likely because translated viral proteins are unable to transfer from the ER to LDs. Copyright © 2017 the Author(s). Published by PNAS.

The small heat shock protein, HSP30, is associated with aggresome-like inclusion bodies in proteasomal inhibitor-, arsenite-, and cadmium-treated Xenopus kidney cells.

PubMed

Khan, Saad; Khamis, Imran; Heikkila, John J

2015-11-01

In the present study, treatment of Xenopus laevis A6 kidney epithelial cells with the proteasomal inhibitor, MG132, or the environmental toxicants, sodium arsenite or cadmium chloride, induced the accumulation of the small heat shock protein, HSP30, in total and in both soluble and insoluble protein fractions. Immunocytochemical analysis revealed the presence of relatively large HSP30 structures primarily in the perinuclear region of the cytoplasm. All three of the stressors promoted the formation of aggresome-like inclusion bodies as determined by immunocytochemistry and laser scanning confocal microscopy using a ProteoStat aggresome dye and additional aggresomal markers, namely, anti-γ-tubulin and anti-vimentin antibodies. Further analysis revealed that HSP30 co-localized with these aggresome-like inclusion bodies. In most cells, HSP30 was found to envelope or occur within these structures. Finally, we show that treatment of cells with withaferin A, a steroidal lactone with anti-inflammatory, anti-tumor, and proteasomal inhibitor properties, also induced HSP30 accumulation that co-localized with aggresome-like inclusion bodies. It is possible that proteasomal inhibitor or metal/metalloid-induced formation of aggresome-like inclusion bodies may sequester toxic protein aggregates until they can be degraded. While the role of HSP30 in these aggresome-like structures is not known, it is possible that they may be involved in various aspects of aggresome-like inclusion body formation or transport. Copyright © 2015 Elsevier Inc. All rights reserved.

Kinesin-microtubule interactions during gliding assays under magnetic force

NASA Astrophysics Data System (ADS)

Fallesen, Todd L.

Conventional kinesin is a motor protein capable of converting the chemical energy of ATP into mechanical work. In the cell, this is used to actively transport vesicles through the intracellular matrix. The relationship between the velocity of a single kinesin, as it works against an increasing opposing load, has been well studied. The relationship between the velocity of a cargo being moved by multiple kinesin motors against an opposing load has not been established. A major difficulty in determining the force-velocity relationship for multiple motors is determining the number of motors that are moving a cargo against an opposing load. Here I report on a novel method for detaching microtubules bound to a superparamagnetic bead from kinesin anchor points in an upside down gliding assay using a uniform magnetic field perpendicular to the direction of microtubule travel. The anchor points are presumably kinesin motors bound to the surface which microtubules are gliding over. Determining the distance between anchor points, d, allows the calculation of the average number of kinesins, n, that are moving a microtubule. It is possible to calculate the fraction of motors able to move microtubules as well, which is determined to be ˜ 5%. Using a uniform magnetic field parallel to the direction of microtubule travel, it is possible to impart a uniform magnetic field on a microtubule bound to a superparamagnetic bead. We are able to decrease the average velocity of microtubules driven by multiple kinesin motors moving against an opposing force. Using the average number of kinesins on a microtubule, we estimate that there are an average 2-7 kinesins acting against the opposing force. By fitting Gaussians to the smoothed distributions of microtubule velocities acting against an opposing force, multiple velocities are seen, presumably for n, n-1, n-2, etc motors acting together. When these velocities are scaled for the average number of motors on a microtubule, the force

The bipolar assembly domain of the mitotic motor kinesin-5

PubMed Central

Acar, Seyda; Carlson, David B.; Budamagunta, Madhu S.; Yarov-Yarovoy, Vladimir; Correia, John J.; Niñonuevo, Milady R.; Jia, Weitao; Tao, Li; Leary, Julie A.; Voss, John C.; Evans, James E.; Scholey, Jonathan M.

2013-01-01

An outstanding unresolved question is how does the mitotic spindle utilize microtubules and mitotic motors to coordinate accurate chromosome segregation during mitosis? This process depends upon the mitotic motor, kinesin-5, whose unique bipolar architecture, with pairs of motor domains lying at opposite ends of a central rod, allows it to crosslink microtubules within the mitotic spindle and to coordinate their relative sliding during spindle assembly, maintenance and elongation. The structural basis of kinesin-5’s bipolarity is, however, unknown, as protein asymmetry has so far precluded its crystallization. Here we use electron microscopy of single molecules of kinesin-5 and its subfragments, combined with hydrodynamic analysis plus mass spectrometry, circular dichroism and site-directed spin label electron paramagnetic resonance spectroscopy, to show how a staggered antiparallel coiled-coil ‘BASS’ (bipolar assembly) domain directs the assembly of four kinesin-5 polypeptides into bipolar minifilaments. PMID:23299893

Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

NASA Technical Reports Server (NTRS)

Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

2002-01-01

Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

Comprehensive comparative analysis of kinesins in photosynthetic eukaryotes

PubMed Central

Richardson, Dale N; Simmons, Mark P; Reddy, Anireddy SN

2006-01-01

Background Kinesins, a superfamily of molecular motors, use microtubules as tracks and transport diverse cellular cargoes. All kinesins contain a highly conserved ~350 amino acid motor domain. Previous analysis of the completed genome sequence of one flowering plant (Arabidopsis) has resulted in identification of 61 kinesins. The recent completion of genome sequencing of several photosynthetic and non-photosynthetic eukaryotes that belong to divergent lineages offers a unique opportunity to conduct a comprehensive comparative analysis of kinesins in plant and non-plant systems and infer their evolutionary relationships. Results We used the kinesin motor domain to identify kinesins in the completed genome sequences of 19 species, including 13 newly sequenced genomes. Among the newly analyzed genomes, six represent photosynthetic eukaryotes. A total of 529 kinesins was used to perform comprehensive analysis of kinesins and to construct gene trees using the Bayesian and parsimony approaches. The previously recognized 14 families of kinesins are resolved as distinct lineages in our inferred gene tree. At least three of the 14 kinesin families are not represented in flowering plants. Chlamydomonas, a green alga that is part of the lineage that includes land plants, has at least nine of the 14 known kinesin families. Seven of ten families present in flowering plants are represented in Chlamydomonas, indicating that these families were retained in both the flowering-plant and green algae lineages. Conclusion The increase in the number of kinesins in flowering plants is due to vast expansion of the Kinesin-14 and Kinesin-7 families. The Kinesin-14 family, which typically contains a C-terminal motor, has many plant kinesins that have the motor domain at the N terminus, in the middle, or the C terminus. Several domains in kinesins are present exclusively either in plant or animal lineages. Addition of novel domains to kinesins in lineage-specific groups contributed to the

Paxillin and embryonic PolyAdenylation Binding Protein (ePABP) engage to regulate androgen-dependent Xenopus laevis oocyte maturation - A model of kinase-dependent regulation of protein expression.

PubMed

Miedlich, Susanne U; Taya, Manisha; Young, Melissa Rasar; Hammes, Stephen R

2017-06-15

Steroid-triggered Xenopus laevis oocyte maturation is an elegant physiologic model of nongenomic steroid signaling, as it proceeds completely independent of transcription. We previously demonstrated that androgens are the main physiologic stimulator of oocyte maturation in Xenopus oocytes, and that the adaptor protein paxillin plays a crucial role in mediating this process through a positive feedback loop in which paxillin first enhances Mos protein translation, ensued by Erk2 activation and Erk-dependent phosphorylation of paxillin on serine residues. Phosphoserine-paxillin then further augments Mos protein translation and downstream Erk2 activation, resulting in meiotic progression. We hypothesized that paxillin enhances Mos translation by interacting with embryonic PolyAdenylation Binding Protein (ePABP) on polyadenylated Mos mRNA. Knockdown of ePABP phenocopied paxillin knockdown, with reduced Mos protein expression, Erk2 and Cdk1 activation, as well as oocyte maturation. In both Xenopus oocytes and mammalian cells (HEK-293), paxillin and ePABP constitutively interacted. Testosterone (Xenopus) or EGF (HEK-293) augmented ePABP-paxillin binding, as well as ePABP binding to Mos mRNA (Xenopus), in an Erk-dependent fashion. Thus, ePABP and paxillin work together in an Erk-dependent fashion to enhance Mos protein translation and promote oocyte maturation. Copyright © 2017 Elsevier B.V. All rights reserved.

A fluid membrane enhances the velocity of cargo transport by small teams of kinesin-1

NASA Astrophysics Data System (ADS)

Li, Qiaochu; Tseng, Kuo-Fu; King, Stephen J.; Qiu, Weihong; Xu, Jing

2018-03-01

Kinesin-1 (hereafter referred to as kinesin) is a major microtubule-based motor protein for plus-end-directed intracellular transport in live cells. While the single-molecule functions of kinesin are well characterized, the physiologically relevant transport of membranous cargos by small teams of kinesins remains poorly understood. A key experimental challenge remains in the quantitative control of the number of motors driving transport. Here we utilized "motile fraction" to overcome this challenge and experimentally accessed transport by a single kinesin through the physiologically relevant transport by a small team of kinesins. We used a fluid lipid bilayer to model the cellular membrane in vitro and employed optical trapping to quantify the transport of membrane-enclosed cargos versus traditional membrane-free cargos under identical conditions. We found that coupling motors via a fluid membrane significantly enhances the velocity of cargo transport by small teams of kinesins. Importantly, enclosing a cargo in a fluid lipid membrane did not impact single-kinesin transport, indicating that membrane-dependent velocity enhancement for team-based transport arises from altered interactions between kinesins. Our study demonstrates that membrane-based coupling between motors is a key determinant of kinesin-based transport. Enhanced velocity may be critical for fast delivery of cargos in live cells.

Modelling of internal architecture of kinesin nanomotor as a machine language.

PubMed

Khataee, H R; Ibrahim, M Y

2012-09-01

Kinesin is a protein-based natural nanomotor that transports molecular cargoes within cells by walking along microtubules. Kinesin nanomotor is considered as a bio-nanoagent which is able to sense the cell through its sensors (i.e. its heads and tail), make the decision internally and perform actions on the cell through its actuator (i.e. its motor domain). The study maps the agent-based architectural model of internal decision-making process of kinesin nanomotor to a machine language using an automata algorithm. The applied automata algorithm receives the internal agent-based architectural model of kinesin nanomotor as a deterministic finite automaton (DFA) model and generates a regular machine language. The generated regular machine language was acceptable by the architectural DFA model of the nanomotor and also in good agreement with its natural behaviour. The internal agent-based architectural model of kinesin nanomotor indicates the degree of autonomy and intelligence of the nanomotor interactions with its cell. Thus, our developed regular machine language can model the degree of autonomy and intelligence of kinesin nanomotor interactions with its cell as a language. Modelling of internal architectures of autonomous and intelligent bio-nanosystems as machine languages can lay the foundation towards the concept of bio-nanoswarms and next phases of the bio-nanorobotic systems development.

Expression of the adhesion G protein-coupled receptor A2 (adgra2) during Xenopus laevis development.

PubMed

Seigfried, Franziska A; Dietmann, Petra; Kühl, Michael; Kühl, Susanne J

2018-06-01

The adhesion G protein-coupled receptor A2 (Adgra2) is a seven transmembrane receptor that has been described to be a regulator for angiogenesis in mice. Furthermore, the zebrafish ouchless mutant is unable to develop dorsal root ganglia through a disrupted trafficking of Adgra2. Besides RNA sequencing data, nothing is reported about Adgra2 in the south African crawled frog Xenopus laevis. In this study, we investigated for the first time the spatio-temporal expression of adgra2 during early Xenopus embryogenesis in detail. In silico approaches showed that the genomic adgra2 region as well as the Adgra2 protein sequence is highly conserved among different species including Xenopus. RT-PCR experiments confirmed that embryonic adgra2 expression is primarily detected at the beginning of neurulation and is then present throughout the whole Xenopus embryogenesis until stage 42. Whole mount in situ hybridization approaches visualized adgra2 expression in many tissues during Xenopus embryogenesis such as the cardiovascular system including the heart, the migrating neural crest cells and the developing eye including the periocular mesenchyme. Our results indicate a role of Adgra2 for embryogenesis and are a good starting point for further functional studies during early vertebrate development. Copyright © 2018 Elsevier B.V. All rights reserved.

Motion of kinesin in a viscoelastic medium

NASA Astrophysics Data System (ADS)

Knoops, Gert; Vanderzande, Carlo

2018-05-01

Kinesin is a molecular motor that transports cargo along microtubules. The results of many in vitro experiments on kinesin-1 are described by kinetic models in which one transition corresponds to the forward motion and subsequent binding of the tethered motor head. We argue that in a viscoelastic medium like the cytosol of a cell this step is not Markov and has to be described by a nonexponential waiting time distribution. We introduce a semi-Markov kinetic model for kinesin that takes this effect into account. We calculate, for arbitrary waiting time distributions, the moment generating function of the number of steps made, and determine from this the average velocity and the diffusion constant of the motor. We illustrate our results for the case of a waiting time distribution that is Weibull. We find that for realistic parameter values, viscoelasticity decreases the velocity and the diffusion constant, but increases the randomness (or Fano factor).

Microscale transport and sorting by kinesin molecular motors.

PubMed

Jia, Lili; Moorjani, Samira G; Jackson, Thomas N; Hancock, William O

2004-03-01

As biomolecular detection systems shrink in size, there is an increasing demand for systems that transport and position materials at micron- and nanoscale dimensions. Our goal is to combine cellular transport machinery-kinesin molecular motors and microtubules-with integrated optoelectronics into a hybrid biological/engineered microdevice that will bind, transport, and detect specific proteins, DNA/RNA molecules, viruses, or cells. For microscale transport, 1.5 microm deep channels were created with SU-8 photoresist on glass, kinesin motors adsorbed to the bottom of the channels, and the channel walls used to bend and redirect microtubules moving over the immobilized motors. Novel channel geometries were investigated as a means to redirect and sort microtubules moving in these channels. We show that DC and AC electric fields are sufficient to transport microtubules in solution, establishing an approach for redirecting microtubules moving in channels. Finally, we inverted the geometry to demonstrate that kinesins can transport gold nanowires along surface immobilized microtubules, providing a model for nanoscale directed assembly.

Ski represses bone morphogenic protein signaling in Xenopus and mammalian cells

PubMed Central

Wang, Wei; Mariani, Francesca V.; Harland, Richard M.; Luo, Kunxin

2000-01-01

The bone morphogenic proteins (BMPs) play important roles in vertebrate development. In Xenopus, BMPs act as epidermal inducers and also as negative regulators of neurogenesis. Antagonism of BMP signaling results in neuralization. BMPs signal through the cell-surface receptors and downstream Smad molecules. Upon stimulation with BMP, Smad1, Smad5, and Smad8 are phosphorylated by the activated BMP receptors, form a complex with Smad4, and translocate into the nucleus, where they regulate the expression of BMP target genes. Here, we show that the Ski oncoprotein can block BMP signaling and the expression of BMP-responsive genes in both Xenopus and mammalian cells by directly interacting with and repressing the activity of BMP-specific Smad complexes. This ability to antagonize BMP signaling results in neuralization by Ski in the Xenopus embryo and blocking of osteoblast differentiation of murine W-20-17 cells. Thus, Ski is able to repress the activity of all receptor-associated Smads and may regulate vertebrate development by modulating the signaling activity of transforming growth factor-β family members. PMID:11121043

Ski represses bone morphogenic protein signaling in Xenopus and mammalian cells.

PubMed

Wang, W; Mariani, F V; Harland, R M; Luo, K

2000-12-19

The bone morphogenic proteins (BMPs) play important roles in vertebrate development. In Xenopus, BMPs act as epidermal inducers and also as negative regulators of neurogenesis. Antagonism of BMP signaling results in neuralization. BMPs signal through the cell-surface receptors and downstream Smad molecules. Upon stimulation with BMP, Smad1, Smad5, and Smad8 are phosphorylated by the activated BMP receptors, form a complex with Smad4, and translocate into the nucleus, where they regulate the expression of BMP target genes. Here, we show that the Ski oncoprotein can block BMP signaling and the expression of BMP-responsive genes in both Xenopus and mammalian cells by directly interacting with and repressing the activity of BMP-specific Smad complexes. This ability to antagonize BMP signaling results in neuralization by Ski in the Xenopus embryo and blocking of osteoblast differentiation of murine W-20-17 cells. Thus, Ski is able to repress the activity of all receptor-associated Smads and may regulate vertebrate development by modulating the signaling activity of transforming growth factor-beta family members.

Regulation of microtubule-based transport by MAP4

PubMed Central

Semenova, Irina; Ikeda, Kazuho; Resaul, Karim; Kraikivski, Pavel; Aguiar, Mike; Gygi, Steven; Zaliapin, Ilya; Cowan, Ann; Rodionov, Vladimir

2014-01-01

Microtubule (MT)-based transport of organelles driven by the opposing MT motors kinesins and dynein is tightly regulated in cells, but the underlying molecular mechanisms remain largely unknown. Here we tested the regulation of MT transport by the ubiquitous protein MAP4 using Xenopus melanophores as an experimental system. In these cells, pigment granules (melanosomes) move along MTs to the cell center (aggregation) or to the periphery (dispersion) by means of cytoplasmic dynein and kinesin-2, respectively. We found that aggregation signals induced phosphorylation of threonine residues in the MT-binding domain of the Xenopus MAP4 (XMAP4), thus decreasing binding of this protein to MTs. Overexpression of XMAP4 inhibited pigment aggregation by shortening dynein-dependent MT runs of melanosomes, whereas removal of XMAP4 from MTs reduced the length of kinesin-2–dependent runs and suppressed pigment dispersion. We hypothesize that binding of XMAP4 to MTs negatively regulates dynein-dependent movement of melanosomes and positively regulates kinesin-2–based movement. Phosphorylation during pigment aggregation reduces binding of XMAP4 to MTs, thus increasing dynein-dependent and decreasing kinesin-2–dependent motility of melanosomes, which stimulates their accumulation in the cell center, whereas dephosphorylation of XMAP4 during dispersion has an opposite effect. PMID:25143402

Protein 4.1 and its interaction with other cytoskeletal proteins in Xenopus laevis oogenesis.

PubMed

Carotenuto, Rosa; Petrucci, Tamara C; Correas, Isabel; Vaccaro, Maria C; De Marco, Nadia; Dale, Brian; Wilding, Martin

2009-06-01

In human red blood cells, protein 4.1 (4.1R) is an 80-kDa polypeptide that stabilizes the spectrin-actin network and anchors it to the plasma membrane. In non-erythroid cells there is a great variety of 4.1R isoforms, mainly generated by alternative pre-mRNA splicing, which localize at various intracellular sites, including the nucleus. We studied protein 4.1R distribution in relation to beta-spectrin, actin and cytokeratin during Xenopus oogenesis. Immunoprecipitation experiments indicate that at least two isoforms of protein 4.1R are present in Xenopus laevis oocytes: a 56-kDa form in the cytoplasm and a 37-kDa form in the germinal vesicle (GV). Antibodies to beta-spectrin reveal two bands of 239 and 100 kDa in the cytoplasm. Coimmunoprecipitation experiments indicate that both the 37- and 56-kDa isoforms of protein 4.1R associate with the 100-kDa isoform of beta-spectrin. Moreover, the 56-kDa form coimmunoprecipitates with a cytokeratin of the same molecular weight. Confocal immunolocalization shows that protein 4.1R distribution is in the peripheral cytoplasm, in the mitochondrial cloud (MC) and in the GV of previtellogenic oocytes. In the cytoplasm of vitellogenic oocytes, a loose network of fibers stained by the anti-protein 4.1R antibody spreads across the cytoplasm. beta-Spectrin has a similar distribution. Protein 4.1R was found to colocalize with actin in the cortex of oocytes in the form of fluorescent dots. Double immunolocalization of protein 4.1R and cytokeratin depicts two separate networks that overlap throughout the whole cytoplasm. Protein 4.1R filaments partially colocalize with cytokeratin in both the animal and vegetal hemispheres. We hypothesize that protein 4.1R could function as a linker protein between cytokeratin and the actin-based cytoskeleton.

Kinesins and Myosins: Molecular Motors that Coordinate Cellular Functions in Plants.

PubMed

Nebenführ, Andreas; Dixit, Ram

2018-04-29

Kinesins and myosins are motor proteins that can move actively along microtubules and actin filaments, respectively. Plants have evolved a unique set of motors that function as regulators and organizers of the cytoskeleton and as drivers of long-distance transport of various cellular components. Recent progress has established the full complement of motors encoded in plant genomes and has revealed valuable insights into the cellular functions of many kinesin and myosin isoforms. Interestingly, several of the motors were found to functionally connect the two cytoskeletal systems and thereby to coordinate their activities. In this review, we discuss the available genetic, cell biological, and biochemical data for each of the plant kinesin and myosin families from the context of their subcellular mechanism of action as well as their physiological function in the whole plant. We particularly emphasize work that illustrates mechanisms by which kinesins and myosins coordinate the activities of the cytoskeletal system.

FRET measurements of kinesin neck orientation reveal a structural basis for processivity and asymmetry.

PubMed

Martin, Douglas S; Fathi, Reza; Mitchison, Timothy J; Gelles, Jeff

2010-03-23

As the smallest and simplest motor enzymes, kinesins have served as the prototype for understanding the relationship between protein structure and mechanochemical function of enzymes in this class. Conventional kinesin (kinesin-1) is a motor enzyme that transports cargo toward the plus end of microtubules by a processive, asymmetric hand-over-hand mechanism. The coiled-coil neck domain, which connects the two kinesin motor domains, contributes to kinesin processivity (the ability to take many steps in a row) and is proposed to be a key determinant of the asymmetry in the kinesin mechanism. While previous studies have defined the orientation and position of microtubule-bound kinesin motor domains, the disposition of the neck coiled-coil remains uncertain. We determined the neck coiled-coil orientation using a multidonor fluorescence resonance energy transfer (FRET) technique to measure distances between microtubules and bound kinesin molecules. Microtubules were labeled with a new fluorescent taxol donor, TAMRA-X-taxol, and kinesin derivatives with an acceptor fluorophore attached at positions on the motor and neck coiled-coil domains were used to reconstruct the positions and orientations of the domains. FRET measurements to positions on the motor domain were largely consistent with the domain orientation determined in previous studies, validating the technique. Measurements to positions on the neck coiled-coil were inconsistent with a radial orientation and instead demonstrated that the neck coiled-coil is parallel to the microtubule surface. The measured orientation provides a structural explanation for how neck surface residues enhance processivity and suggests a simple hypothesis for the origin of kinesin step asymmetry and "limping."

Electrostatically Biased Binding of Kinesin to Microtubules

PubMed Central

Zheng, Wenjun; Alonso, Maria; Huber, Gary; Dlugosz, Maciej; McCammon, J. Andrew; Cross, Robert A.

2011-01-01

The minimum motor domain of kinesin-1 is a single head. Recent evidence suggests that such minimal motor domains generate force by a biased binding mechanism, in which they preferentially select binding sites on the microtubule that lie ahead in the progress direction of the motor. A specific molecular mechanism for biased binding has, however, so far been lacking. Here we use atomistic Brownian dynamics simulations combined with experimental mutagenesis to show that incoming kinesin heads undergo electrostatically guided diffusion-to-capture by microtubules, and that this produces directionally biased binding. Kinesin-1 heads are initially rotated by the electrostatic field so that their tubulin-binding sites face inwards, and then steered towards a plus-endwards binding site. In tethered kinesin dimers, this bias is amplified. A 3-residue sequence (RAK) in kinesin helix alpha-6 is predicted to be important for electrostatic guidance. Real-world mutagenesis of this sequence powerfully influences kinesin-driven microtubule sliding, with one mutant producing a 5-fold acceleration over wild type. We conclude that electrostatic interactions play an important role in the kinesin stepping mechanism, by biasing the diffusional association of kinesin with microtubules. PMID:22140358

A method for multiprotein assembly in cells reveals independent action of kinesins in complex

PubMed Central

Norris, Stephen R.; Soppina, Virupakshi; Dizaji, Aslan S.; Schimert, Kristin I.; Sept, David; Cai, Dawen; Sivaramakrishnan, Sivaraj

2014-01-01

Teams of processive molecular motors are critical for intracellular transport and organization, yet coordination between motors remains poorly understood. Here, we develop a system using protein components to generate assemblies of defined spacing and composition inside cells. This system is applicable to studying macromolecular complexes in the context of cell signaling, motility, and intracellular trafficking. We use the system to study the emergent behavior of kinesin motors in teams. We find that two kinesin motors in complex act independently (do not help or hinder each other) and can alternate their activities. For complexes containing a slow kinesin-1 and fast kinesin-3 motor, the slow motor dominates motility in vitro but the fast motor can dominate on certain subpopulations of microtubules in cells. Both motors showed dynamic interactions with the complex, suggesting that motor–cargo linkages are sensitive to forces applied by the motors. We conclude that kinesin motors in complex act independently in a manner regulated by the microtubule track. PMID:25365993

Measuring collective transport by defined numbers of processive and nonprocessive kinesin motors.

PubMed

Furuta, Ken'ya; Furuta, Akane; Toyoshima, Yoko Y; Amino, Misako; Oiwa, Kazuhiro; Kojima, Hiroaki

2013-01-08

Intracellular transport is thought to be achieved by teams of motor proteins bound to a cargo. However, the coordination within a team remains poorly understood as a result of the experimental difficulty in controlling the number and composition of motors. Here, we developed an experimental system that links together defined numbers of motors with defined spacing on a DNA scaffold. By using this system, we linked multiple molecules of two different types of kinesin motors, processive kinesin-1 or nonprocessive Ncd (kinesin-14), in vitro. Both types of kinesins markedly increased their processivities with motor number. Remarkably, despite the poor processivity of individual Ncd motors, the coupling of two Ncd motors enables processive movement for more than 1 μm along microtubules (MTs). This improvement was further enhanced with decreasing spacing between motors. Force measurements revealed that the force generated by groups of Ncd is additive when two to four Ncd motors work together, which is much larger than that generated by single motors. By contrast, the force of multiple kinesin-1s depends only weakly on motor number. Numerical simulations and single-molecule unbinding measurements suggest that this additive nature of the force exerted by Ncd relies on fast MT binding kinetics and the large drag force of individual Ncd motors. These features would enable small groups of Ncd motors to crosslink MTs while rapidly modulating their force by forming clusters. Thus, our experimental system may provide a platform to study the collective behavior of motor proteins from the bottom up.

Mechanical splitting of microtubules into protofilament bundles by surface-bound kinesin-1

DOE PAGES

VanDelinder, Virginia; Adams, Peter G.; Bachand, George D.

2016-12-21

The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices. In these systems, phenomena such as molecular wear and fracture into shorter MTs have been reported due the mechanical forces applied on the MT during transport. In the present work, we show that MTs can be split longitudinally into protofilament bundles (PFBs) by the work performed by surface-bound kinesin motors. We examine the properties of these PFBs using several techniques (e.g., fluorescence microscopy, SEM, AFM), and show that the PFBs continuemore » to be mobile on the surface and display very high curvature compared to MT. Further, higher surface density of kinesin motors and shorter kinesin-surface tethers promote PFB formation, whereas modifying MT with GMPCPP or higher paclitaxel concentrations did not affect PFB formation.« less

Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains

PubMed Central

1988-01-01

We have prepared and characterized seven mouse monoclonal antibodies (SUK 1-7) to the 130-kD heavy chain of sea urchin egg kinesin. On immunoblots, SUK 3 and SUK 4 cross-reacted with Drosophila embryo 116- kD heavy chains, and SUK 4, SUK 5, SUK 6, and SUK 7 bound to the 120-kD heavy chains of bovine brain kinesin. Three out of seven monoclonal antikinesins (SUK 4, SUK 6, and SUK 7) caused a dose-dependent inhibition of sea urchin egg kinesin-induced microtubule translocation, whereas the other four monoclonal antibodies had no detectable effect on this motility. The inhibitory monoclonal antibodies (SUK 4, SUK 6, and SUK 7) appear to bind to spatially related sites on an ATP- sensitive microtubule binding 45-kD chymotryptic fragment of the 130-kD heavy chain, whereas SUK 2 binds to a spatially distinct site. None of the monoclonal antikinesins inhibited the microtubule activated MgATPase activity of kinesin, suggesting that SUK 4, SUK 6, and SUK 7 uncouple this MgATPase activity from motility. PMID:2974459

Isolation and Expression Profile of the Ca2+-Activated Chloride Channel-like Membrane Protein 6 Gene in Xenopus laevis

PubMed Central

Lee, Ra Mi; Ryu, Rae Hyung; Jeong, Seong Won; Oh, Soo Jin; Huang, Hue; Han, Jin Soo; Lee, Chi Ho; Lee, C. Justin; Jan, Lily Yeh

2011-01-01

To clone the first anion channel from Xenopus laevis (X. laevis), we isolated a calcium-activated chloride channel (CLCA)-like membrane protein 6 gene (CMP6) in X. laevis. As a first step in gene isolation, an expressed sequence tags database was screened to find the partial cDNA fragment. A putative partial cDNA sequence was obtained by comparison with rat CLCAs identified in our laboratory. First stranded cDNA was synthesized by reverse transcription polymerase-chain reaction (RT-PCR) using a specific primer designed for the target cDNA. Repeating the 5' and 3' rapid amplification of cDNA ends, full-length cDNA was constructed from the cDNA pool. The full-length CMP6 cDNA completed via 5'- and 3'-RACE was 2,940 bp long and had an open reading frame (ORF) of 940 amino acids. The predicted 940 polypeptides have four major transmembrane domains and showed about 50% identity with that of rat brain CLCAs in our previously published data. Semi-quantification analysis revealed that CMP6 was most abundantly expressed in small intestine, colon and liver. However, all tissues except small intestine, colon and liver had undetectable levels. This result became more credible after we did real-time PCR quantification for the target gene. In view of all CLCA studies focused on human or murine channels, this finding suggests a hypothetical protein as an ion channel, an X. laevis CLCA. PMID:21826170

The kinesin KIF9 and reggie/flotillin proteins regulate matrix degradation by macrophage podosomes

PubMed Central

Cornfine, Susanne; Himmel, Mirko; Kopp, Petra; el Azzouzi, Karim; Wiesner, Christiane; Krüger, Marcus; Rudel, Thomas; Linder, Stefan

2011-01-01

Podosomes are actin-based matrix contacts in a variety of cell types, most notably monocytic cells, and are characterized by their ability to lyse extracellular matrix material. Besides their dependence on actin regulation, podosomes are also influenced by microtubules and microtubule-dependent transport processes. Here we describe a novel role for KIF9, a previously little-characterized member of the kinesin motor family, in the regulation of podosomes in primary human macrophages. We find that small interfering RNA (siRNA)/short-hairpin RNA–induced knockdown of KIF9 significantly affects both numbers and matrix degradation of podosomes. Overexpression and microinjection experiments reveal that the unique C-terminal region of KIF9 is crucial for these effects, presumably through binding of specific interactors. Indeed, we further identify reggie-1/flotillin-2, a signaling mediator between intracellular vesicles and the cell periphery, as an interactor of the KIF9 C-terminus. Reggie-1 dynamically colocalizes with KIF9 in living cells, and, consistent with KIF9-mediated effects, siRNA-induced knockdown of reggies/flotillins significantly impairs matrix degradation by podosomes. In sum, we identify the kinesin KIF9 and reggie/flotillin proteins as novel regulators of macrophage podosomes and show that their interaction is critical for the matrix-degrading ability of these structures. PMID:21119006

The yeast kinesin-5 Cin8 interacts with the microtubule in a noncanonical manner

PubMed Central

Bell, Kayla M.; Cha, Hyo Keun; Sindelar, Charles V.; Cochran, Jared C.

2017-01-01

Kinesin motors play central roles in establishing and maintaining the mitotic spindle during cell division. Unlike most other kinesins, Cin8, a kinesin-5 motor in Saccharomyces cerevisiae, can move bidirectionally along microtubules, switching directionality according to biochemical conditions, a behavior that remains largely unexplained. To this end, we used biochemical rate and equilibrium constant measurements as well as cryo-electron microscopy methodologies to investigate the microtubule interactions of the Cin8 motor domain. These experiments unexpectedly revealed that, whereas Cin8 ATPase kinetics fell within measured ranges for kinesins (especially kinesin-5 proteins), approximately four motors can bind each αβ-tubulin dimer within the microtubule lattice. This result contrasted with those observations on other known kinesins, which can bind only a single “canonical” site per tubulin dimer. Competition assays with human kinesin-5 (Eg5) only partially abrogated this behavior, indicating that Cin8 binds microtubules not only at the canonical site, but also one or more separate (“noncanonical”) sites. Moreover, we found that deleting the large, class-specific insert in the microtubule-binding loop 8 reverts Cin8 to one motor per αβ-tubulin in the microtubule. The novel microtubule-binding mode of Cin8 identified here provides a potential explanation for Cin8 clustering along microtubules and potentially may contribute to the mechanism for direction reversal. PMID:28701465

In Vitro Motility of Liver Connexin Vesicles along Microtubules Utilizes Kinesin Motors*

PubMed Central

Fort, Alfredo G.; Murray, John W.; Dandachi, Nadine; Davidson, Michael W.; Dermietzel, Rolf; Wolkoff, Allan W.; Spray, David C.

2011-01-01

Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin- and tubulin-based systems remain unclear. We have observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 μm/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 μm of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 μm ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4–0.5 μm/s, which was inhibited with 1 mm of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 μm vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32. PMID:21536677

Chlamydomonas Kinesin-II–dependent Intraflagellar Transport (IFT): IFT Particles Contain Proteins Required for Ciliary Assembly in Caenorhabditis elegans Sensory Neurons

PubMed Central

Cole, Douglas G.; Diener, Dennis R.; Himelblau, Amy L.; Beech, Peter L.; Fuster, Jason C.; Rosenbaum, Joel L.

1998-01-01

We previously described a kinesin-dependent movement of particles in the flagella of Chlamydomonas reinhardtii called intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519–5523). When IFT is inhibited by inactivation of a kinesin, FLA10, in the temperature-sensitive mutant, fla10, existing flagella resorb and new flagella cannot be assembled. We report here that: (a) the IFT-associated FLA10 protein is a subunit of a heterotrimeric kinesin; (b) IFT particles are composed of 15 polypeptides comprising two large complexes; (c) the FLA10 kinesin-II and IFT particle polypeptides, in addition to being found in flagella, are highly concentrated around the flagellar basal bodies; and, (d) mutations affecting homologs of two of the IFT particle polypeptides in Caenorhabditis elegans result in defects in the sensory cilia located on the dendritic processes of sensory neurons. In the accompanying report by Pazour, G.J., C.G. Wilkerson, and G.B. Witman (1998. J. Cell Biol. 141:979–992), a Chlamydomonas mutant (fla14) is described in which only the retrograde transport of IFT particles is disrupted, resulting in assembly-defective flagella filled with an excess of IFT particles. This microtubule- dependent transport process, IFT, defined by mutants in both the anterograde (fla10) and retrograde (fla14) transport of isolable particles, is probably essential for the maintenance and assembly of all eukaryotic motile flagella and nonmotile sensory cilia. PMID:9585417

Kinesin expands and stabilizes the GDP-microtubule lattice

NASA Astrophysics Data System (ADS)

Peet, Daniel R.; Burroughs, Nigel J.; Cross, Robert A.

2018-05-01

Kinesin-1 is a nanoscale molecular motor that walks towards the fast-growing (plus) ends of microtubules, hauling molecular cargo to specific reaction sites in cells. Kinesin-driven transport is central to the self-organization of eukaryotic cells and shows great promise as a tool for nano-engineering1. Recent work hints that kinesin may also play a role in modulating the stability of its microtubule track, both in vitro2,3 and in vivo4, but the results are conflicting5-7 and the mechanisms are unclear. Here, we report a new dimension to the kinesin-microtubule interaction, whereby strong-binding state (adenosine triphosphate (ATP)-bound and apo) kinesin-1 motor domains inhibit the shrinkage of guanosine diphosphate (GDP) microtubules by up to two orders of magnitude and expand their lattice spacing by 1.6%. Our data reveal an unexpected mechanism by which the mechanochemical cycles of kinesin and tubulin interlock, and so allow motile kinesins to influence the structure, stability and mechanics of their microtubule track.

Purification of a novel, nucleoplasmin-like protein from somatic nuclei.

PubMed Central

Cotten, M; Chalkley, R

1987-01-01

We have purified a nucleoplasmin-like protein from the nuclei of somatic Xenopus laevis cells. This protein possesses a number of the distinctive features of nucleoplasmin isolated from oocytes or unfertilized eggs. The protein is recognized by both monoclonal and polyclonal antisera raised against egg nucleoplasmin. The protein has an oligomeric structure, which must be heated in SDS to completely dissociate, is acidic, phosphorylated and efficiently promotes the in vitro formation of chromatin. We have partially characterized this novel protein and because of its resemblance to nucleoplasmin isolated from oocytes or unfertilized eggs we have named this protein nucleoplasmin S. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:3443097

Chromosome congression by kinesin-5 motor-mediated disassembly of longer kinetochore microtubules

PubMed Central

Gardner, Melissa K; Bouck, David C.; Paliulis, Leocadia V.; Meehl, Janet B.; O’Toole, Eileen T.; Haase, Julian; Soubry, Adelheid; Joglekar, Ajit P.; Winey, Mark; Salmon, Edward D.; Bloom, Kerry; Odde, David J.

2008-01-01

Summary During mitosis, sister chromatids congress to the spindle equator and are subsequently segregated via attachment to dynamic kinetochore microtubule (kMT) plus-ends. A major question is how kMT plus-end assembly is spatially regulated to achieve chromosome congression. Here we find in budding yeast that the widely-conserved kinesin-5 sliding motor proteins, Cin8p and Kip1p, mediate chromosome congression by suppressing kMT plus-end assembly of longer kMTs. Of the two, Cin8p is the major effector and its activity requires a functional motor domain. In contrast, the depolymerizing kinesin-8 motor Kip3p plays a minor role in spatial regulation of yeast kMT assembly. Our analysis identified a model where kinesin-5 motors bind to kMTs, move to kMT plus ends, and upon arrival at a growing plus-end promote net kMT plus-end disassembly. In conclusion, we find that length-dependent control of net kMT assembly by kinesin-5 motors yields a simple and stable self-organizing mechanism for chromosome congression. PMID:19041752

Effect of fuel concentration on cargo transport by a team of Kinesin motors

NASA Astrophysics Data System (ADS)

Takshak, Anjneya; Mishra, Nirvantosh; Kulkarni, Aditi; Kunwar, Ambarish

2017-02-01

Eukaryotic cells employ specialized proteins called molecular motors for transporting organelles and vesicles from one location to another in a regulated and directed manner. These molecular motors often work collectively in a team while transporting cargos. Molecular motors use cytoplasmic ATP as fuel, which is hydrolyzed to generate mechanical force. While the effect of ATP concentration on cargo transport by single Kinesin motor function is well understood, it is still unexplored, both theoretically and experimentally, how ATP concentration would affect cargo transport by a team of Kinesin motors. For instance, how does fuel concentration affect the travel distances and travel velocities of cargo? How cooperativity of Kinesin motors engaged on a cargo is affected by ATP concentration? To answer these questions, here we develop mechano-chemical models of cargo transport by a team of Kinesin motors. To develop these models we use experimentally-constrained mechano-chemical model of a single Kinesin motor as well as earlier developed mean-field and stochastic models of load sharing for cargo transport. Thus, our new models for cargo transport by a team of Kinesin motors include fuel concentration explicitly, which was not considered in earlier models. We make several interesting predictions which can be tested experimentally. For instance, the travel distances of cargos are very large at limited ATP concentrations in spite of very small travel velocity. Velocities of cargos driven by multiple Kinesin have a Michaelis-Menten dependence on ATP concentration. Similarly, cooperativity among the engaged Kinesin motors on the cargo shows a Michaelis-Menten type dependence, which attains a maximum value near physiological ATP concentrations. Our new results can be potentially useful in controlling artificial nano-molecular shuttles precisely for targeted delivery in various nano-technological applications.

Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins

PubMed Central

Gillespie, Peter J.; Gambus, Agnieszka; Blow, J. Julian

2012-01-01

The use of cell-free extracts prepared from eggs of the South African clawed toad, Xenopus laevis, has led to many important discoveries in cell cycle research. These egg extracts recapitulate the key nuclear transitions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. DNA added to the extract is first assembled into a nucleus and is then efficiently replicated. Progression of the extract into mitosis then allows the separation of paired sister chromatids. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. In this article we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei for the study of DNA replication in vitro. We also detail how DNA replication can be quantified in this system. In addition, we describe methods for isolating chromatin and chromatin-bound protein complexes from egg extracts. These recently developed and revised techniques provide a practical starting point for investigating the function of proteins involved in DNA replication. PMID:22521908

KinG Is a Plant-Specific Kinesin That Regulates Both Intra- and Intercellular Movement of SHORT-ROOT.

PubMed

Spiegelman, Ziv; Lee, Chin-Mei; Gallagher, Kimberly L

2018-01-01

Both endogenous plant proteins and viral movement proteins associate with microtubules to promote their movement through plasmodesmata. The association of viral movement proteins with microtubules facilitates the formation of virus-associated replication complexes, which are required for the amplification and subsequent spread of the virus. However, the role of microtubules in the intercellular movement of plant proteins is less clear. Here we show that the SHORT-ROOT (SHR) protein, which moves between cells in the root to regulate root radial patterning, interacts with a type-14 kinesin, KINESIN G (KinG). KinG is a calponin homology domain kinesin that directly interacts with the SHR-binding protein SIEL (SHR-INTERACING EMBRYONIC LETHAL) and localizes to both microtubules and actin. Since SIEL and SHR associate with endosomes, we suggest that KinG serves as a linker between SIEL, SHR, and the plant cytoskeleton. Loss of KinG function results in a decrease in the intercellular movement of SHR and an increase in the sensitivity of SHR movement to treatment with oryzalin. Examination of SHR and KinG localization and dynamics in live cells suggests that KinG is a nonmotile kinesin that promotes the pausing of SHR-associated endosomes. We suggest a model in which interaction of KinG with SHR allows for the formation of stable movement complexes that facilitate the cell-to-cell transport of SHR. © 2018 American Society of Plant Biologists. All Rights Reserved.

Activity of the kinesin spindle protein inhibitor ispinesib (SB-715992) in models of breast cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Purcell, James W; Davis, Jefferson; Reddy, Mamatha

2009-06-10

Ispinesib (SB-715992) is a potent inhibitor of kinesin spindle protein (KSP), a kinesin motor protein essential for the formation of a bipolar mitotic spindle and cell cycle progression through mitosis. Clinical studies of ispinesib have demonstrated a 9% response rate in patients with locally advanced or metastatic breast cancer, and a favorable safety profile without significant neurotoxicities, gastrointestinal toxicities or hair loss. To better understand the potential of ispinesib in the treatment of breast cancer we explored the activity of ispinesib alone and in combination several therapies approved for the treatment of breast cancer. We measured the ispinesib sensitivity andmore » pharmacodynamic response of breast cancer cell lines representative of various subtypes in vitro and as xenografts in vivo, and tested the ability of ispinesib to enhance the anti-tumor activity of approved therapies. In vitro, ispinesib displayed broad anti-proliferative activity against a panel of 53 breast cell-lines. In vivo, ispinesib produced regressions in each of five breast cancer models, and tumor free survivors in three of these models. The effects of ispinesib treatment on pharmacodynamic markers of mitosis and apoptosis were examined in vitro and in vivo, revealing a greater increase in both mitotic and apoptotic markers in the MDA-MB-468 model than in the less sensitive BT-474 model. In vivo, ispinesib enhanced the anti-tumor activity of trastuzumab, lapatinib, doxorubicin, and capecitabine, and exhibited activity comparable to paclitaxel and ixabepilone. These findings support further clinical exploration of KSP inhibitors for the treatment of breast cancer.« less

Recycling of Kinesin-1 Motors by Diffusion after Transport

PubMed Central

Blasius, T. Lynne; Reed, Nathan; Slepchenko, Boris M.; Verhey, Kristen J.

2013-01-01

Kinesin motors drive the long-distance anterograde transport of cellular components along microtubule tracks. Kinesin-dependent transport plays a critical role in neurogenesis and neuronal function due to the large distance separating the soma and nerve terminal. The fate of kinesin motors after delivery of their cargoes is unknown but has been postulated to involve degradation at the nerve terminal, recycling via retrograde motors, and/or recycling via diffusion. We set out to test these models concerning the fate of kinesin-1 motors after completion of transport in neuronal cells. We find that kinesin-1 motors are neither degraded nor returned by retrograde motors. By combining mathematical modeling and experimental analysis, we propose a model in which the distribution and recycling of kinesin-1 motors fits a “loose bucket brigade” where individual motors alter between periods of active transport and free diffusion within neuronal processes. These results suggest that individual kinesin-1 motors are utilized for multiple rounds of transport. PMID:24098765

The function of Xenopus Bloom's syndrome protein homolog (xBLM) in DNA replication

PubMed Central

Liao, Shuren; Graham, Jeanine; Yan, Hong

2000-01-01

The Bloom's syndrome gene (BLM) plays a pivotal role in the maintenance of genomic stability in somatic cells. It encodes a DNA helicase (BLM) of the RecQ family, but the exact function of BLM remains elusive. To study this question, we have cloned the BLM homolog of the frog Xenopus laevis (xBLM) and have raised antibodies to it. Immunodepletion of xBLM from a Xenopus egg extract severely inhibits the replication of DNA in reconstituted nuclei. Moreover, the inhibition can be rescued by the addition of the recombinant xBLM protein. These results provide the first direct evidence that BLM plays an important role in DNA replication, suggesting that Bloom's syndrome may be the consequence of defective DNA replication. PMID:11040210

Molecular characterization of myelin protein zero in Xenopus laevis peripheral nerve

NASA Astrophysics Data System (ADS)

Xie, Bo; Luo, Xiaoyang; Zhao, Cheng; Priest, Christina Marie; Chan, Shiu-Yung; O'Connor, Peter B.; Kirschner, Daniel A.; Costello, Catherine E.

2007-12-01

Myelin protein zero (P0), a glycosylated single-pass transmembrane protein, is essential in the formation and maintenance of peripheral nervous system (PNS) compact myelin. P0 in Xenopus (xP0) exists primarily as a dimeric form that remains stable after various physical and chemical treatments. In exploring the nature of the interactions underlying the dimer stability, we found that xP0 dimer dissociated into monomer during continuous elution gel electrophoresis and conventional SDS-PAGE, indicating that the dimer is stabilized by non-covalent interactions. Furthermore, as some of the gel-purified monomer re-associated into dimer on SDS-PAGE gels, there is likely a dynamic equilibrium between xP0 dimer and monomer in vivo. Because the carbohydrate and fatty acyl moieties may be crucial for the adhesion role of P0, we used sensitive mass spectrometry approaches to elucidate the detailed N-glycosylation and S-acylation profiles of xP0. Asn92 was determined to be the single, fully-occupied glycosylation site of xP0, and a total of 12 glycans was detected that exhibited new structural features compared with those observed from P0 in other species: (1) the neutral glycans were composed mainly of high mannose and hybrid types; (2) 5 of 12 were acidic glycans, among which three were sialylated and the other two were sulfated; (3) none of the glycans had core fucosylation; and (4) no glucuronic acid, hence no HNK-1 epitope, was detected. The drastically different carbohydrate structures observed here support the concept of the species-specific variation in N-glycosylation of P0. Cys152 was found to be acylated with stearoyl (C18:0), whereas palmitoyl (C16:0) is the corresponding predominant fatty acyl group on P0 from higher vertebrates. We propose that the unique glycosylation and acylation patterns of Xenopus P0 may underlie its unusual dimerization behavior. Our results should shed light on the understanding of the phylogenetic development of P0's adhesion role in PNS

Kinesin and Dynein Mechanics: Measurement Methods and Research Applications.

PubMed

Abraham, Zachary; Hawley, Emma; Hayosh, Daniel; Webster-Wood, Victoria A; Akkus, Ozan

2018-02-01

Motor proteins play critical roles in the normal function of cells and proper development of organisms. Among motor proteins, failings in the normal function of two types of proteins, kinesin and dynein, have been shown to lead many pathologies, including neurodegenerative diseases and cancers. As such, it is critical to researchers to understand the underlying mechanics and behaviors of these proteins, not only to shed light on how failures may lead to disease, but also to guide research toward novel treatment and nano-engineering solutions. To this end, many experimental techniques have been developed to measure the force and motility capabilities of these proteins. This review will (a) discuss such techniques, specifically microscopy, atomic force microscopy (AFM), optical trapping, and magnetic tweezers, and (b) the resulting nanomechanical properties of motor protein functions such as stalling force, velocity, and dependence on adenosine triphosophate (ATP) concentrations will be comparatively discussed. Additionally, this review will highlight the clinical importance of these proteins. Furthermore, as the understanding of the structure and function of motor proteins improves, novel applications are emerging in the field. Specifically, researchers have begun to modify the structure of existing proteins, thereby engineering novel elements to alter and improve native motor protein function, or even allow the motor proteins to perform entirely new tasks as parts of nanomachines. Kinesin and dynein are vital elements for the proper function of cells. While many exciting experiments have shed light on their function, mechanics, and applications, additional research is needed to completely understand their behavior.

The Mechanochemical Cycle of Mammalian Kinesin-2 KIF3A/B under Load.

PubMed

Andreasson, Johan O L; Shastry, Shankar; Hancock, William O; Block, Steven M

2015-05-04

The response of motor proteins to external loads underlies their ability to work in teams and determines the net speed and directionality of cargo transport. The mammalian kinesin-2, KIF3A/B, is a heterotrimeric motor involved in intraflagellar transport and vesicle motility in neurons. Bidirectional cargo transport is known to result from the opposing activities of KIF3A/B and dynein bound to the same cargo, but the load-dependent properties of kinesin-2 are poorly understood. We used a feedback-controlled optical trap to probe the velocity, run length, and unbinding kinetics of mouse KIF3A/B under various loads and nucleotide conditions. The kinesin-2 motor velocity is less sensitive than kinesin-1 to external forces, but its processivity diminishes steeply with load, and the motor was observed occasionally to slip and reattach. Each motor domain was characterized by studying homodimeric constructs, and a global fit to the data resulted in a comprehensive pathway that quantifies the principal force-dependent kinetic transitions. The properties of the KIF3A/B heterodimer are intermediate between the two homodimers, and the distinct load-dependent behavior is attributable to the properties of the motor domains and not to the neck linkers or the coiled-coil stalk. We conclude that the force-dependent movement of KIF3A/B differs significantly from conventional kinesin-1. Against opposing dynein forces, KIF3A/B motors are predicted to rapidly unbind and rebind, resulting in qualitatively different transport behavior from kinesin-1. Copyright © 2015 Elsevier Ltd. All rights reserved.

The mechanochemical cycle of mammalian kinesin-2 KIF3A/B under load

PubMed Central

Andreasson, Johan O.L.; Shastry, Shankar; Hancock, William O.; Block, Steven M.

2015-01-01

Summary The response of motor proteins to external loads underlies their ability to work in teams and determines the net speed and directionality of cargo transport. The mammalian kinesin-2, KIF3A/B, is a heterotrimeric motor involved in intraflagellar transport and vesicle motility in neurons. Bidirectional cargo transport is known to result from the opposing activities of KIF3A/B and dynein bound to the same cargo, but the load-dependent properties of kinesin-2 are poorly understood. We used a feedback-controlled optical trap to probe the velocity, run length and unbinding kinetics of mouse KIF3A/B under various loads and nucleotide conditions. The kinesin-2 motor velocity is less sensitive than kinesin-1 to external forces, but its processivity diminishes steeply with load, and the motor was observed occasionally to slip and reattach. Each motor domain was characterized by studying homodimeric constructs, and a global fit to the data resulted in a comprehensive pathway that quantifies the principal force-dependent kinetic transitions. The properties of the KIF3A/B heterodimer are intermediate between the two homodimers, and the distinct load-dependent behavior is attributable to the properties of the motor domains, and not to the neck-linkers or the coiled-coil stalk. We conclude that the force-dependent movement of KIF3A/B differs significantly from conventional kinesin-1. Against opposing dynein forces, KIF3A/B motors are predicted to rapidly unbind and rebind, resulting in qualitatively different transport behavior from kinesin-1. PMID:25866395

Mutation of the MAP kinase DYF-5 affects docking and undocking of kinesin-2 motors and reduces their speed in the cilia of Caenorhabditis elegans

PubMed Central

Burghoorn, Jan; Dekkers, Martijn P. J.; Rademakers, Suzanne; de Jong, Ton; Willemsen, Rob; Jansen, Gert

2007-01-01

In the cilia of the nematode Caenorhabditis elegans, anterograde intraflagellar transport (IFT) is mediated by two kinesin-2 complexes, kinesin II and OSM-3 kinesin. These complexes function together in the cilia middle segments, whereas OSM-3 alone mediates transport in the distal segments. Not much is known about the mechanisms that compartmentalize the kinesin-2 complexes or how transport by both kinesins is coordinated. Here, we identify DYF-5, a conserved MAP kinase that plays a role in these processes. Fluorescence microscopy and EM revealed that the cilia of dyf-5 loss-of-function (lf) animals are elongated and are not properly aligned into the amphid channel. Some cilia do enter the amphid channel, but the distal ends of these cilia show accumulation of proteins. Consistent with these observations, we found that six IFT proteins accumulate in the cilia of dyf-5(lf) mutants. In addition, using genetic analyses and live imaging to measure the motility of IFT proteins, we show that dyf-5 is required to restrict kinesin II to the cilia middle segments. Finally, we show that, in dyf-5(lf) mutants, OSM-3 moves at a reduced speed and is not attached to IFT particles. We propose that DYF-5 plays a role in the undocking of kinesin II from IFT particles and in the docking of OSM-3 onto IFT particles. PMID:17420466

Interaction with a kinesin-2 tail propels choline acetyltransferase flow towards synapse

PubMed Central

Sadananda, Aparna; Hamid, Runa; Doodhi, Harinath; Ghosal, Debnath; Girotra, Mukul; Jana, Swadhin Chandra; Ray, Krishanu

2012-01-01

Bulk flow constitutes a substantial part of the slow transport of soluble proteins in axons. Though the underlying mechanism is unclear, evidences indicate that intermittent, kinesin based movement of large protein-aggregates aids this process. Choline acetyl-transferase (ChAT), a soluble enzyme catalyzing acetylcholine synthesis, propagates towards synapse at an intermediate, slow rate. The presynaptic enrichment of ChAT requires heterotrimeric kinesin-2, comprising KLP64D, KLP68D and DmKAP, in Drosophila. Here, we show that the bulk flow of a recombinant Green Fluorescent Protein-tagged ChAT (GFP::ChAT), in Drosophila axons, lacks particulate features. It occurs for a brief period during the larval stages. In addition, both the endogenous ChAT and GFP::ChAT directly bind to the KLP64D tail, which is essential for the GFP::ChAT entry and anterograde flow in axon. These evidences suggest that a direct interaction with motor proteins could regulate the bulk flow of soluble proteins, and thus establish their asymmetric distribution. PMID:22486887

Ligand- and structure-based in silico studies to identify kinesin spindle protein (KSP) inhibitors as potential anticancer agents.

PubMed

Balakumar, Chandrasekaran; Ramesh, Muthusamy; Tham, Chuin Lean; Khathi, Samukelisiwe Pretty; Kozielski, Frank; Srinivasulu, Cherukupalli; Hampannavar, Girish A; Sayyad, Nisar; Soliman, Mahmoud E; Karpoormath, Rajshekhar

2017-11-29

Kinesin spindle protein (KSP) belongs to the kinesin superfamily of microtubule-based motor proteins. KSP is responsible for the establishment of the bipolar mitotic spindle which mediates cell division. Inhibition of KSP expedites the blockade of the normal cell cycle during mitosis through the generation of monoastral MT arrays that finally cause apoptotic cell death. As KSP is highly expressed in proliferating/cancer cells, it has gained considerable attention as a potential drug target for cancer chemotherapy. Therefore, this study envisaged to design novel KSP inhibitors by employing computational techniques/tools such as pharmacophore modelling, virtual database screening, molecular docking and molecular dynamics. Initially, the pharmacophore models were generated from the data-set of highly potent KSP inhibitors and the pharmacophore models were validated against in house test set ligands. The validated pharmacophore model was then taken for database screening (Maybridge and ChemBridge) to yield hits, which were further filtered for their drug-likeliness. The potential hits retrieved from virtual database screening were docked using CDOCKER to identify the ligand binding landscape. The top-ranked hits obtained from molecular docking were progressed to molecular dynamics (AMBER) simulations to deduce the ligand binding affinity. This study identified MB-41570 and CB-10358 as potential hits and evaluated these experimentally using in vitro KSP ATPase inhibition assays.

Kinesin spindle protein (KSP) inhibitors. Part 4: Structure-based design of 5-alkylamino-3,5-diaryl-4,5-dihydropyrazoles as potent, water-soluble inhibitors of the mitotic kinesin KSP.

PubMed

Cox, Christopher D; Torrent, Maricel; Breslin, Michael J; Mariano, Brenda J; Whitman, David B; Coleman, Paul J; Buser, Carolyn A; Walsh, Eileen S; Hamilton, Kelly; Schaber, Michael D; Lobell, Robert B; Tao, Weikang; South, Vicki J; Kohl, Nancy E; Yan, Youwei; Kuo, Lawrence C; Prueksaritanont, Thomayant; Slaughter, Donald E; Li, Chunze; Mahan, Elizabeth; Lu, Bing; Hartman, George D

2006-06-15

Molecular modeling in combination with X-ray crystallographic information was employed to identify a region of the kinesin spindle protein (KSP) binding site not fully utilized by our first generation inhibitors. We discovered that by appending a propylamine substituent at the C5 carbon of a dihydropyrazole core, we could effectively fill this unoccupied region of space and engage in a hydrogen-bonding interaction with the enzyme backbone. This change led to a second generation compound with increased potency, a 400-fold enhancement in aqueous solubility at pH 4, and improved dog pharmacokinetics relative to the first generation compound.

An Improved Optical Tweezers Assay for Measuring the Force Generation of Single Kinesin Molecules

PubMed Central

Nicholas, Matthew P.; Rao, Lu; Gennerich, Arne

2014-01-01

Numerous microtubule-associated molecular motors, including several kinesins and cytoplasmic dynein, produce opposing forces that regulate spindle and chromosome positioning during mitosis. The motility and force generation of these motors are therefore critical to normal cell division, and dysfunction of these processes may contribute to human disease. Optical tweezers provide a powerful method for studying the nanometer motility and piconewton force generation of single motor proteins in vitro. Using kinesin-1 as a prototype, we present a set of step-by-step, optimized protocols for expressing a kinesin construct (K560-GFP) in Escherichia coli, purifying it, and studying its force generation in an optical tweezers microscope. We also provide detailed instructions on proper alignment and calibration of an optical trapping microscope. These methods provide a foundation for a variety of similar experiments. PMID:24633799

Microtubule defects influence kinesin-based transport in vitro.

NASA Astrophysics Data System (ADS)

Xu, Jing

Microtubules are protein polymers that form ``molecular highways'' for long-range transport within living cells. Molecular motors actively step along microtubules to shuttle cellular materials between the nucleus and the cell periphery; this transport is critical for the survival and health of all eukaryotic cells. Structural defects in microtubules exist, but whether these defects impact molecular motor-based transport remains unknown. Here, we report a new, to our knowledge, approach that allowed us to directly investigate the impact of such defects. Using a modified optical-trapping method, we examined the group function of a major molecular motor, conventional kinesin, when transporting cargos along individual microtubules. We found that microtubule defects influence kinesin-based transport in vitro. The effects depend on motor number: cargos driven by a few motors tended to unbind prematurely from the microtubule, whereas cargos driven by more motors tended to pause. To our knowledge, our study provides the first direct link between microtubule defects and kinesin function. The effects uncovered in our study may have physiological relevance in vivo. Supported by the UC Merced (to J.X.), NIH (NS048501 to S.J.K.), NSF (EF-1038697 to A.G.), and the James S. McDonnell Foundation (to A.G.). Work carried out at the Aspen Center for Physics was supported by NSF Grant PHY-1066293.

Lack of association between the Trp719Arg polymorphism in kinesin-like protein 6 and coronary artery disease in 19 case-control studies

PubMed Central

Assimes, Themistocles L; Hólm, Hilma; Kathiresan, Sekar; Reilly, Muredach P; Thorleifsson, Gudmar; Voight, Benjamin F; Erdmann, Jeanette; Willenborg, Christina; Vaidya, Dhananjay; Xie, Changchun; Patterson, Chris C; Morgan, Thomas M; Burnett, Mary Susan; Li, Mingyao; Hlatky, Mark A; Knowles, Joshua W; Thompson, John R; Absher, Devin; Iribarren, Carlos; Go, Alan; Fortmann, Stephen P; Sidney, Stephen; Risch, Neil; Tang, Hua; Myers, Richard M; Berger, Klaus; Stoll, Monika; Shah, Svati H.; Thorgeirsson, Gudmundur; Andersen, Karl; Havulinna, Aki S; Herrera, J. Enrique; Faraday, Nauder; Kim, Yoonhee; Kral, Brian G.; Mathias, Rasika; Ruczinski, Ingo; Suktitipat, Bhoom; Wilson, Alexander F; Yanek, Lisa R.; Becker, Lewis C; Linsel-Nitschke, Patrick; Lieb, Wolfgang; König, Inke R; Hengstenberg, Christian; Fischer, Marcus; Stark, Klaus; Reinhard, Wibke; Winogradow, Janina; Grassl, Martina; Grosshennig, Anika; Preuss, Michael; Eifert, Sandra; Schreiber, Stefan; Wichmann, H-Erich; Meisinger, Christa; Yee, Jean; Friedlander, Yechiel; Do, Ron; Meigs, James B; Williams, Gordon; Nathan, David M; MacRae, Calum A; Qu, Liming; Wilensky, Robert L; Matthai, William H.; Qasim, Atif N; Hakonarson, Hakon; Pichard, Augusto D; Kent, Kenneth M; Satler, Lowell; Lindsay, Joseph M; Waksman, Ron; Knouff, Christopher W; Waterworth, Dawn M; Walker, Max C; Mooser, Vincent; Marrugat, Jaume; Lucas, Gavin; Subirana, Isaac; Sala, Joan; Ramos, Rafael; Martinelli, Nicola; Olivieri, Oliviero; Trabetti, Elisabetta; Malerba, Giovanni; Pignatti, Pier Franco; Guiducci, Candace; Mirel, Daniel; Parkin, Melissa; Hirschhorn, Joel N; Asselta, Rosanna; Duga, Stefano; Musunuru, Kiran; Daly, Mark J; Purcell, Shaun; Braund, Peter S; Wright, Benjamin J; Balmforth, Anthony J; Ball, Stephen G; Ouwehand, Willem H; Deloukas, Panos; Scholz, Michael; Cambien, Francois; Huge, Andreas; Scheffold, Thomas; Salomaa, Veikko; Girelli, Domenico; Granger, Christopher B.; Peltonen, Leena; McKeown, Pascal P; Altshuler, David; Melander, Olle; Devaney, Joseph M; Epstein, Stephen E; Rader, Daniel J; Elosua, Roberto; Engert, James C; Anand, Sonia S; Hall, Alistair S; Ziegler, Andreas; O’Donnell, Christopher J; Spertus, John A; Siscovick, David; Schwartz, Stephen M; Becker, Diane; Thorsteinsdottir, Unnur; Stefansson, Kari; Schunkert, Heribert; Samani, Nilesh J; Quertermous, Thomas

2011-01-01

Objectives We sought to replicate the association between the kinesin-like protein 6 (KIF6) Trp719Arg polymorphism (rs20455) and clinical coronary artery disease (CAD). Background Recent prospective studies suggest that carriers of the 719Arg allele in KIF6 are at increased risk of clinical CAD compared with non-carriers. Methods The KIF6 Trp719Arg polymorphism (rs20455) was genotyped in nineteen case-control studies of non-fatal CAD either as part of a genome-wide association study or in a formal attempt to replicate the initial positive reports. Results Over 17 000 cases and 39 000 controls of European descent as well as a modest number of South Asians, African Americans, Hispanics, East Asians, and admixed cases and controls were successfully genotyped. None of the nineteen studies demonstrated an increased risk of CAD in carriers of the 719Arg allele compared with non-carriers. Regression analyses and fixed effect meta-analyses ruled out with high degree of confidence an increase of ≥2% in the risk of CAD among European 719Arg carriers. We also observed no increase in the risk of CAD among 719Arg carriers in the subset of Europeans with early onset disease (<50 years of age for males and <60 years for females) compared with similarly aged controls as well as all non-European subgroups. Conclusions The KIF6 Trp719Arg polymorphism was not associated with the risk of clinical CAD in this large replication study. PMID:20933357

A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast

PubMed Central

Yukawa, Masashi; Kawakami, Tomoki; Okazaki, Masaki; Kume, Kazunori; Tang, Ngang Heok; Toda, Takashi

2017-01-01

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end–directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end–directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly. PMID:29021344

Importin-β Directly Regulates the Motor Activity and Turnover of a Kinesin-4.

PubMed

Ganguly, Anindya; DeMott, Logan; Zhu, Chuanmei; McClosky, Daniel D; Anderson, Charles T; Dixit, Ram

2018-03-12

Spatiotemporal regulation of kinesins is essential for microtubule-dependent intracellular transport. In plants, cell wall deposition depends on the FRA1 kinesin, whose abundance and motility are tightly controlled to match cellular growth rate. Here, we show that an importin-β, IMB4, regulates FRA1 activity in a developmental manner. IMB4 physically interacts with a PY motif in the FRA1 motor domain and inhibits its motility by preventing microtubule binding, while also protecting FRA1 against proteasome-mediated degradation, thus providing a mechanism to couple the motility and stability of FRA1. This regulatory mechanism is likely to be broadly applicable, based on the conservation of the PY motif in the motor domains of plant and animal kinesins and the direct interaction of multiple plant kinesins with IMB4. Together, our data establish IMB4 as a multi-functional regulator of FRA1 and reveal a mechanism for how plants control the magnitude of cargo transport needed for cell wall assembly. Copyright © 2018 Elsevier Inc. All rights reserved.

Molecular sorting by electrical steering of microtubules in kinesin-coated channels.

PubMed

van den Heuvel, Martin G L; de Graaff, Martijn P; Dekker, Cees

2006-05-12

Integration of biomolecular motors in nanoengineered structures raises the intriguing possibility of manipulating materials on nanometer scales. We have managed to integrate kinesin motor proteins in closed submicron channels and to realize active electrical control of the direction of individual kinesin-propelled microtubule filaments at Y junctions. Using this technique, we demonstrate molecular sorting of differently labeled microtubules. We attribute the steering of microtubules to electric field-induced bending of the leading tip. From measurements of the orientation-dependent electrophoretic motion of individual, freely suspended microtubules, we estimate the net applied force on the tip to be in the picoNewton range and we infer an effective charge of 12 e- per tubulin dimer under physiological conditions.

A novel assay reveals preferential binding between Rabs, kinesins, and specific endosomal subpopulations

PubMed Central

Bentley, Marvin; Decker, Helena; Luisi, Julie

2015-01-01

Identifying the proteins that regulate vesicle trafficking is a fundamental problem in cell biology. In this paper, we introduce a new assay that involves the expression of an FKBP12-rapamycin–binding domain–tagged candidate vesicle-binding protein, which can be inducibly linked to dynein or kinesin. Vesicles can be labeled by any convenient method. If the candidate protein binds the labeled vesicles, addition of the linker drug results in a predictable, highly distinctive change in vesicle localization. This assay generates robust and easily interpretable results that provide direct experimental evidence of binding between a candidate protein and the vesicle population of interest. We used this approach to compare the binding of Kinesin-3 family members with different endosomal populations. We found that KIF13A and KIF13B bind preferentially to early endosomes and that KIF1A and KIF1Bβ bind preferentially to late endosomes and lysosomes. This assay may have broad utility for identifying the trafficking proteins that bind to different vesicle populations. PMID:25624392

The Genome of the Western Clawed Frog Xenopus tropicalis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hellsten, Uffe; Harland, Richard M.; Gilchrist, Michael J.

2009-10-01

The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes over 20,000 protein-coding genes, including orthologs of at least 1,700 human disease genes. Over a million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like other tetrapods, the genome contains gene deserts enriched for conserved non-coding elements. The genome exhibits remarkable shared synteny with humanmore » and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.« less

A Structural Perspective on the Dynamics of Kinesin Motors

PubMed Central

Hyeon, Changbong; Onuchic, José N.

2011-01-01

Despite significant fluctuation under thermal noise, biological machines in cells perform their tasks with exquisite precision. Using molecular simulation of a coarse-grained model and theoretical arguments, we envisaged how kinesin, a prototype of biological machines, generates force and regulates its dynamics to sustain persistent motor action. A structure-based model, which can be versatile in adapting its structure to external stresses while maintaining its native fold, was employed to account for several features of kinesin dynamics along the biochemical cycle. This analysis complements our current understandings of kinesin dynamics and connections to experiments. We propose a thermodynamic cycle for kinesin that emphasizes the mechanical and regulatory role of the neck linker and clarify issues related to the motor directionality, and the difference between the external stalling force and the internal tension responsible for the head-head coordination. The comparison between the thermodynamic cycle of kinesin and macroscopic heat engines highlights the importance of structural change as the source of work production in biomolecular machines. PMID:22261064

Cloning of noggin gene from hydra and analysis of its functional conservation using Xenopus laevis embryos.

PubMed

Chandramore, Kalpana; Ito, Yuzuro; Takahashi, Shuji; Asashima, Makoto; Ghaskadbi, Surendra

2010-01-01

Hydra, a member of phylum Cnidaria that arose early in evolution, is endowed with a defined axis, organized nervous system, and active behavior. It is a powerful model system for the elucidation of evolution of developmental mechanisms in animals. Here, we describe the identification and cloning of noggin-like gene from hydra. Noggin is a secreted protein involved at multiple stages of vertebrate embryonic development including neural induction and is known to exert its effects by inhibiting the bone morphogenetic protein (BMP)-signaling pathway. Sequence analysis revealed that hydra Noggin shows considerable similarity with its orthologs at the amino acid level. When microinjected in the early Xenopus embryos, hydra noggin mRNA induced a secondary axis in 100% of the injected embryos, demonstrating functional conservation of hydra noggin in vertebrates. This was further confirmed by the partial rescue of Xenopus embryos by hydra noggin mRNA from UV-induced ventralization. By using animal cap assay in Xenopus embryos, we demonstrate that these effects of hydra noggin in Xenopus embryos are because of inhibition of BMP signaling by Noggin. Our data indicate that BMP/Noggin antagonism predates the bilaterian divergence and is conserved during the evolution.

Isthmin is a novel secreted protein expressed as part of the Fgf-8 synexpression group in the Xenopus midbrain-hindbrain organizer.

PubMed

Pera, Edgar M; Kim, James I; Martinez, Sarah L; Brechner, Mariel; Li, Su Yu; Wessely, Oliver; De Robertis, E M

2002-08-01

Patterning of the central nervous system is regulated by a signaling center located at the midbrain-hindbrain boundary (MHB), or isthmus organizer. Fibroblast growth factors secreted from the MHB are required and sufficient to direct the ordered growth and regionalization of the midbrain and anterior hindbrain. In an unbiased secretion cloning screen of Xenopus gastrula embryos we identified a novel gene, which we designated as Isthmin (xIsm) due to its prominent expression at the MHB. xIsm encodes a secreted protein of 449 amino acids containing one copy of the thrombospondin type 1 repeat (TSR). We also found orthologous Isthmin genes in human (hIsm) and mouse (mIsm), as well as a gene encoding an Isthmin-like human unknown protein (hIsm-l). The conservation of a unique carboxy-terminal region between hIsm and hIsm-l suggests that Isthmin is the founding member of a new family of secreted proteins. xIsm was strongly expressed maternally in the Xenopus egg and showed zygotic expression in the ventral blastopore lip, notochord, and MHB. Additional expression domains were detected in neural crest, ear vesicle, and developing blood islands. Interestingly, xIsm was co-expressed with Fibroblast growth factor-8 (xFgf-8) at multiple sites including the MHB, indicating that these two genes are part of a synexpression group which also includes sprouty and sef homologs.

Alp7/TACC recruits kinesin-8–PP1 to the Ndc80 kinetochore protein for timely mitotic progression and chromosome movement

PubMed Central

Tang, Ngang Heok; Toda, Takashi

2015-01-01

ABSTRACT Upon establishment of proper kinetochore–microtubule attachment, the spindle assembly checkpoint (SAC) must be silenced to allow onset of anaphase, which is when sister chromatids segregate equally to two daughter cells. However, how proper kinetochore–microtubule attachment leads to timely anaphase onset remains elusive. Furthermore, the molecular mechanisms of chromosome movement during anaphase A remain unclear. In this study, we show that the fission yeast Alp7/TACC protein recruits a protein complex consisting of the kinesin-8 (Klp5–Klp6) and protein phosphatase 1 (PP1) to the kinetochore upon kinetochore–microtubule attachment. Accumulation of this complex at the kinetochore, on the one hand, facilitates SAC inactivation through PP1, and, on the other hand, accelerates polewards chromosome movement driven by the Klp5–Klp6 motor. We identified an alp7 mutant that had specific defects in binding to the Klp5–Klp6–PP1 complex but with normal localisation to the microtubule and kinetochore. Consistent with our proposition, this mutant shows delayed anaphase onset and decelerated chromosome movement during anaphase A. We propose that the recruitment of kinesin-8–PP1 to the kinetochore through Alp7/TACC interaction plays a crucial role in regulation of timely mitotic progression and chromosome movement during anaphase A. PMID:25472718

A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast.

PubMed

Yukawa, Masashi; Kawakami, Tomoki; Okazaki, Masaki; Kume, Kazunori; Tang, Ngang Heok; Toda, Takashi

2017-12-01

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end-directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end-directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly. © 2017 Yukawa et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

A Molecular atlas of Xenopus respiratory system development.

PubMed

Rankin, Scott A; Thi Tran, Hong; Wlizla, Marcin; Mancini, Pamela; Shifley, Emily T; Bloor, Sean D; Han, Lu; Vleminckx, Kris; Wert, Susan E; Zorn, Aaron M

2015-01-01

Respiratory system development is regulated by a complex series of endoderm-mesoderm interactions that are not fully understood. Recently Xenopus has emerged as an alternative model to investigate early respiratory system development, but the extent to which the morphogenesis and molecular pathways involved are conserved between Xenopus and mammals has not been systematically documented. In this study, we provide a histological and molecular atlas of Xenopus respiratory system development, focusing on Nkx2.1+ respiratory cell fate specification in the developing foregut. We document the expression patterns of Wnt/β-catenin, fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) signaling components in the foregut and show that the molecular mechanisms of respiratory lineage induction are remarkably conserved between Xenopus and mice. Finally, using several functional experiments we refine the epistatic relationships among FGF, Wnt, and BMP signaling in early Xenopus respiratory system development. We demonstrate that Xenopus trachea and lung development, before metamorphosis, is comparable at the cellular and molecular levels to embryonic stages of mouse respiratory system development between embryonic days 8.5 and 10.5. This molecular atlas provides a fundamental starting point for further studies using Xenopus as a model to define the conserved genetic programs controlling early respiratory system development. © 2014 Wiley Periodicals, Inc.

A universal pathway for kinesin stepping.

PubMed

Clancy, Bason E; Behnke-Parks, William M; Andreasson, Johan O L; Rosenfeld, Steven S; Block, Steven M

2011-08-14

Kinesin-1 is an ATP-driven, processive motor that transports cargo along microtubules in a tightly regulated stepping cycle. Efficient gating mechanisms ensure that the sequence of kinetic events proceeds in the proper order, generating a large number of successive reaction cycles. To study gating, we created two mutant constructs with extended neck-linkers and measured their properties using single-molecule optical trapping and ensemble fluorescence techniques. Owing to a reduction in the inter-head tension, the constructs access an otherwise rarely populated conformational state in which both motor heads remain bound to the microtubule. ATP-dependent, processive backstepping and futile hydrolysis were observed under moderate hindering loads. On the basis of measurements, we formulated a comprehensive model for kinesin motion that incorporates reaction pathways for both forward and backward stepping. In addition to inter-head tension, we found that neck-linker orientation is also responsible for ensuring gating in kinesin.

Accumulation of Cytoplasmic Dynein and Dynactin at Microtubule Plus Ends in Aspergillus nidulans Is Kinesin DependentV⃞

PubMed Central

Zhang, Jun; Li, Shihe; Fischer, Reinhard; Xiang, Xin

2003-01-01

The mechanism(s) by which microtubule plus-end tracking proteins are targeted is unknown. In the filamentous fungus Aspergillus nidulans, both cytoplasmic dynein and NUDF, the homolog of the LIS1 protein, localize to microtubule plus ends as comet-like structures. Herein, we show that NUDM, the p150 subunit of dynactin, also forms dynamic comet-like structures at microtubule plus ends. By examining proteins tagged with green fluorescent protein in different loss-of-function mutants, we demonstrate that dynactin and cytoplasmic dynein require each other for microtubule plus-end accumulation, and the presence of cytoplasmic dynein is also important for NUDF's plus-end accumulation. Interestingly, deletion of NUDF increases the overall accumulation of dynein and dynactin at plus ends, suggesting that NUDF may facilitate minus-end–directed dynein movement. Finally, we demonstrate that a conventional kinesin, KINA, is required for the microtubule plus-end accumulation of cytoplasmic dynein and dynactin, but not of NUDF. PMID:12686603

Discovery of a vezatin-like protein for dynein-mediated early endosome transport

PubMed Central

Yao, Xuanli; Arst, Herbert N.; Wang, Xiangfeng; Xiang, Xin

2015-01-01

Early endosomes are transported bidirectionally by cytoplasmic dynein and kinesin-3, but how the movements are regulated in vivo remains unclear. Here our forward genetic study led to the discovery of VezA, a vezatin-like protein in Aspergillus nidulans, as a factor critical for early endosome distribution. Loss of vezA causes an abnormal accumulation of early endosomes at the hyphal tip, where microtubule plus ends are located. This abnormal accumulation depends on kinesin-3 and is due to a decrease in the frequency but not the speed of dynein-mediated early endosome movement. VezA-GFP signals are enriched at the hypha tip in an actin-dependent manner but are not obviously associated with early endosomes, thus differing from the early endosome association of the cargo adapter HookA (Hook in A. nidulans). On loss of VezA, HookA associates normally with early endosomes, but the interaction between dynein-dynactin and the early-endosome-bound HookA is significantly decreased. However, VezA is not required for linking dynein-dynactin to the cytosolic ∆C-HookA, lacking the cargo-binding C-terminus. These results identify VezA as a novel regulator required for the interaction between dynein and the Hook-bound early endosomes in vivo. PMID:26378255

Loading direction regulates the affinity of ADP for kinesin.

PubMed

Uemura, Sotaro; Ishiwata, Shin'ichi

2003-04-01

Kinesin is an ATP-driven molecular motor that moves processively along a microtubule. Processivity has been explained as a mechanism that involves alternating single- and double-headed binding of kinesin to microtubules coupled to the ATPase cycle of the motor. The internal load imposed between the two bound heads has been proposed to be a key factor regulating the ATPase cycle in each head. Here we show that external load imposed along the direction of motility on a single kinesin molecule enhances the binding affinity of ADP for kinesin, whereas an external load imposed against the direction of motility decreases it. This coupling between loading direction and enzymatic activity is in accord with the idea that the internal load plays a key role in the unidirectional and cooperative movement of processive motors.

Multimotor Transport in a System of Active and Inactive Kinesin-1 Motors

PubMed Central

Scharrel, Lara; Ma, Rui; Schneider, René; Jülicher, Frank; Diez, Stefan

2014-01-01

Long-range directional transport in cells is facilitated by microtubule-based motor proteins. One example is transport in a nerve cell, where small groups of motor proteins, such as kinesins and cytoplasmic dynein, work together to ensure the supply and clearance of cellular material along the axon. Defects in axonal transport have been linked to Alzheimer’s and other neurodegenerative diseases. However, it is not known in detail how multimotor-based cargo transport is impaired if a fraction of the motors are defective. To mimic impaired multimotor transport in vitro, we performed gliding motility assays with varying fractions of active kinesin-1 motors and inactive kinesin-1 motor mutants. We found that impaired transport manifests in multiple motility regimes: 1), a fast-motility regime characterized by gliding at velocities close to the single-molecule velocity of the active motors; 2), a slow-motility regime characterized by gliding at close-to zero velocity or full stopping; and 3), a regime in which fast and slow motilities coexist. Notably, the transition from the fast to the slow regime occurred sharply at a threshold fraction of active motors. Based on single-motor parameters, we developed a stochastic model and a mean-field theoretical description that explain our experimental findings. Our results demonstrate that impaired multimotor transport mostly occurs in an either/or fashion: depending on the ratio of active to inactive motors, transport is either performed at close to full speed or is out of action. PMID:25028878

Homologs of the Xenopus developmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis of Nod-like chitin oligosaccharides during early embryogenesis.

PubMed

Semino, C E; Specht, C A; Raimondi, A; Robbins, P W

1996-05-14

The Xenopus developmental gene DG42 is expressed during early embryonic development, between the midblastula and neurulation stages. The deduced protein sequence of Xenopus DG42 shows similarity to Rhizobium Nod C, Streptococcus Has A, and fungal chitin synthases. Previously, we found that the DG42 protein made in an in vitro transcription/translation system catalyzed synthesis of an array of chitin oligosaccharides. Here we show that cell extracts from early Xenopus and zebrafish embryos also synthesize chitooligosaccharides. cDNA fragments homologous to DG42 from zebrafish and mouse were also cloned and sequenced. Expression of these homologs was similar to that described for Xenopus based on Northern and Western blot analysis. The Xenopus anti-DG42 antibody recognized a 63-kDa protein in extracts from zebrafish embryos that followed a similar developmental expression pattern to that previously described for Xenopus. The chitin oligosaccharide synthase activity found in extracts was inactivated by a specific DG42 antibody; synthesis of hyaluronic acid (HA) was not affected under the conditions tested. Other experiments demonstrate that expression of DG42 under plasmid control in mouse 3T3 cells gives rise to chitooligosaccharide synthase activity without an increase in HA synthase level. A possible relationship between our results and those of other investigators, which show stimulation of HA synthesis by DG42 in mammalian cell culture systems, is provided by structural analyses to be published elsewhere that suggest that chitin oligosaccharides are present at the reducing ends of HA chains. Since in at least one vertebrate system hyaluronic acid formation can be inhibited by a pure chitinase, it seems possible that chitin oligosaccharides serve as primers for hyaluronic acid synthesis.

Kinesin regulation dynamics through cargo delivery, a single molecule investigation

NASA Astrophysics Data System (ADS)

Kovacs, Anthony; Kessler, Jonathan; Lin, Huawen; Dutcher, Susan; Wang, Yan Mei

2015-03-01

Kinesins are microtubule-based motors that deliver cargo to their destinations in a highly regulated manner. Although in recent years numerous regulators of cargo delivery have been identified, the regulation mechanism of kinesin through the cargo delivery and recycling process is not known. By performing single molecule fluorescence imaging measurements in Chlamydomonas flagella, which are 200 nm in diameter, 10 microns in length, and contain 9 sets of microtubule doublets, we tracked the intraflagellar transport (IFT) trains, BBSome cargo, and kinesin-2 motors through the cargo delivery process and determined the aforementioned dynamics. Upon arrival at the microtubule plus end at the flagellar tip, (1) IFT trains and BBSome cargo remain intact, dissociate together from kinesins and microtubules, and diffuse along flagellar membrane for a mean of 2.3 sec before commencing retrograde travel. (2) Kinesin motors remain bound to and diffuse along microtubules for 1.3 sec before dissociating into the flagellar lumen for recycling.

Single Molecule Investigation of Kinesin-1 Motility Using Engineered Microtubule Defects

NASA Astrophysics Data System (ADS)

Gramlich, Michael W.; Conway, Leslie; Liang, Winnie H.; Labastide, Joelle A.; King, Stephen J.; Xu, Jing; Ross, Jennifer L.

2017-03-01

The structure of the microtubule is tightly regulated in cells via a number of microtubule associated proteins and enzymes. Microtubules accumulate structural defects during polymerization, and defect size can further increase under mechanical stresses. Intriguingly, microtubule defects have been shown to be targeted for removal via severing enzymes or self-repair. The cell’s control in defect removal suggests that defects can impact microtubule-based processes, including molecular motor-based intracellular transport. We previously demonstrated that microtubule defects influence cargo transport by multiple kinesin motors. However, mechanistic investigations of the observed effects remained challenging, since defects occur randomly during polymerization and are not directly observable in current motility assays. To overcome this challenge, we used end-to-end annealing to generate defects that are directly observable using standard epi-fluorescence microscopy. We demonstrate that the annealed sites recapitulate the effects of polymerization-derived defects on multiple-motor transport, and thus represent a simple and appropriate model for naturally-occurring defects. We found that single kinesins undergo premature dissociation, but not preferential pausing, at the annealed sites. Our findings provide the first mechanistic insight to how defects impact kinesin-based transport. Preferential dissociation on the single-molecule level has the potential to impair cargo delivery at locations of microtubule defect sites in vivo.

Chromosome and mitotic spindle dynamics in fission yeast kinesin-8 mutants

NASA Astrophysics Data System (ADS)

Crapo, Ammon M.; Gergley, Zachary R.; McIntosh, J. Richard; Betterton, M. D.

2014-03-01

Fission yeast proteins Klp5p and Klp6p are plus-end directed motors of the kinesin-8 family which promote microtubule (MT) depolymerization and also affect chromosome segregation, but the mechanism of these activities is not well understood. Using live-cell time-lapse fluorescence microscopy of fission yeast wild-type (WT) and klp5/6 mutant strains, we quantify and compare the dynamics of kinetochore motion and mitotic spindle length in 3D. In WT cells, the spindle, once formed, remains a consistent size and chromosomes are correctly organized and segregated. In kinesin-8 mutants, spindles undergo large length fluctuations of several microns. Kinetochore motions are also highly fluctuating, with kinetochores frequently moving away from the spindle rather than toward it. We observe transient pushing of chromosomes away from the spindle by as much as 10 microns in distance.

Analysis of a developmentally regulated nuclear localization signal in Xenopus

PubMed Central

1992-01-01

The 289 residue nuclear oncoprotein encoded by the adenovirus 5 Ela gene contains two peptide sequences that behave as nuclear localization signals (NLS). One signal, located at the carboxy terminus, is like many other known NLSs in that it consists of a short stretch of basic residues (KRPRP) and is constitutively active in cells. The second signal resides within an internal 45 residue region of E1a that contains few basic residues or sequences that resemble other known NLSs. Moreover, this internal signal functions in injected Xenopus oocytes, but not in transfected Xenopus A6 cells, suggesting that it could be regulated developmentally (Slavicek et al. 1989. J. Virol. 63:4047). In this study, we show that the activity of this signal is sensitive to ATP depletion in vivo, efficiently directs the import of a 50 kD fusion protein and can compete with the E1a carboxy-terminal NLS for nuclear import. In addition, we have delineated the precise amino acid residues that comprise the second E1a NLS, and have assessed its utilization during Xenopus embryogenesis. Using amino acid deletion and substitution analyses, we show that the signal consists of the sequence FV(X)7-20MXSLXYM(X)4MF. By expressing in Xenopus embryos a truncated E1a protein that contains only the second NLS and by monitoring its cytoplasmic/nuclear distribution during development with indirect immunofluorescence, we find that the second NLS is utilized up to the early neurula stage. In addition, there appears to be a hierarchy among the embryonic germ layers as to when the second NLS becomes nonfunctional. For this reason, we refer to this NLS as the developmentally regulated nuclear localization signal (drNLS). The implications of these findings for early development are discussed. PMID:1387407

Family-specific Kinesin Structures Reveal Neck-linker Length Based on Initiation of the Coiled-coil*

PubMed Central

Phillips, Rebecca K.; Peter, Logan G.; Gilbert, Susan P.

2016-01-01

Kinesin-1, -2, -5, and -7 generate processive hand-over-hand 8-nm steps to transport intracellular cargoes toward the microtubule plus end. This processive motility requires gating mechanisms to coordinate the mechanochemical cycles of the two motor heads to sustain the processive run. A key structural element believed to regulate the degree of processivity is the neck-linker, a short peptide of 12–18 residues, which connects the motor domain to its coiled-coil stalk. Although a shorter neck-linker has been correlated with longer run lengths, the structural data to support this hypothesis have been lacking. To test this hypothesis, seven kinesin structures were determined by x-ray crystallography. Each included the neck-linker motif, followed by helix α7 that constitutes the start of the coiled-coil stalk. In the majority of the structures, the neck-linker length differed from predictions because helix α7, which initiates the coiled-coil, started earlier in the sequence than predicted. A further examination of structures in the Protein Data Bank reveals that there is a great disparity between the predicted and observed starting residues. This suggests that an accurate prediction of the start of a coiled-coil is currently difficult to achieve. These results are significant because they now exclude simple comparisons between members of the kinesin superfamily and add a further layer of complexity when interpreting the results of mutagenesis or protein fusion. They also re-emphasize the need to consider factors beyond the kinesin neck-linker motif when attempting to understand how inter-head communication is tuned to achieve the degree of processivity required for cellular function. PMID:27462072

Mitotic Chromosome Biorientation in Fission Yeast Is Enhanced by Dynein and a Minus-end–directed, Kinesin-like Protein

PubMed Central

Spiridonov, Ilia S.; McIntosh, J. Richard

2007-01-01

Chromosome biorientation, the attachment of sister kinetochores to sister spindle poles, is vitally important for accurate chromosome segregation. We have studied this process by following the congression of pole-proximal kinetochores and their subsequent anaphase segregation in fission yeast cells that carry deletions in any or all of this organism's minus end–directed, microtubule-dependent motors: two related kinesin 14s (Pkl1p and Klp2p) and dynein. None of these deletions abolished biorientation, but fewer chromosomes segregated normally without Pkl1p, and to a lesser degree without dynein, than in wild-type cells. In the absence of Pkl1p, which normally localizes to the spindle and its poles, the checkpoint that monitors chromosome biorientation was defective, leading to frequent precocious anaphase. Ultrastructural analysis of mutant mitotic spindles suggests that Pkl1p contributes to error-free biorientation by promoting normal spindle pole organization, whereas dynein helps to anchor a focused bundle of spindle microtubules at the pole. PMID:17409356

Recent findings and future directions for interpolar mitotic kinesin inhibitors in cancer therapy.

PubMed

Myers, Stephanie M; Collins, Ian

2016-01-01

The kinesin class of microtubule-associated motor proteins present attractive anticancer targets owing to their roles in key functions in dividing cells. Two interpolar mitotic kinesins Eg5 and HSET have opposing motor functions in mitotic spindle assembly with respect to microtubule movement, but both offer opportunities to develop cancer selective therapeutic agents. Here, we summarize the progress to date in developing inhibitors of Eg5 and HSET, with an emphasis on structural biology insights into the binding modes of allosteric inhibitors, compound selectivity and mechanisms of action of different chemical scaffolds. We discuss translation of preclinical studies to clinical experience with Eg5 inhibitors, recent findings on potential resistance mechanisms and explore the implications for future anticancer drug development against these targets.

Recent findings and future directions for interpolar mitotic kinesin inhibitors in cancer therapy

PubMed Central

Myers, Stephanie M.; Collins, Ian

2016-01-01

The kinesin class of microtubule-associated motor proteins present attractive anti-cancer targets owing to their roles in key functions in dividing cells. Two interpolar mitotic kinesins Eg5 and HSET have opposing motor functions in mitotic spindle assembly with respect to microtubule movement, but both offer opportunities to develop cancer selective therapeutic agents. Here, we summarize the progress to date in developing inhibitors of Eg5 and HSET, with an emphasis on structural biology insights into the binding modes of allosteric inhibitors, compound selectivity and mechanisms of action of different chemical scaffolds. We discuss translation of preclinical studies to clinical experience with Eg5 inhibitors, recent findings on potential resistance mechanisms, and explore the implications for future anticancer drug development against these targets. PMID:26976726

Localization of a microtubule organizing center by kinesin motors

NASA Astrophysics Data System (ADS)

Arita, Chikashi; Bosche, Jonas; Lück, Alexander; Santen, Ludger

2017-12-01

Molecular motors are proteins which bind to a polarized cytoskeletal filament and move steadily along it. Molecular motors of the kinesin family move along microtubules (MTs), which are a component of the cytoskeleton. A very processive kinesin motor Kip3p, is known to promote catastrophes and pausing of MT, in particular on cortical contact. These properties play an important role in positioning the mitotic spindle in budding yeast. We present a theoretical approach to positioning of MT networks under confinement. In order to explore a localization mechanism of a microtubule organizing center (MTOC), we introduce an idealized system of two MTs connected by a MTOC. The dynamics of Kip3p is modeled by interacting stochastic particles, which allows us to study the effects of motor-induced depolymerization in a finite volume. We find that localization in the middle of the cavity is realized in a parameter regime where the motor densities on the MTs are increasing with the distance from the MTOC. Localization at an asymmetric position is also possible by tuning model parameters.

Establishment and characterization of Xenopus oviduct cells in primary culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marsh, J.; Tata, J.R.

1987-11-01

Based on previously established procedure of Xenopus hepatocytes, the authors describe tubular oviduct cells in primary culture which continue to secrete substantial quantities of egg jelly for several days, as can be visualized microscopically. Freshly isolated cells exhibited a culture shock response, from which they recovered by the third day in culture. This recovery was characterized by (a) the diminished synthesis of heat shock proteins hsp 70 and hsp 85, (b) the cessation of the drop in number of estrogen receptor, and (c) the enhanced rate of synthesis of cellular and secreted proteins. The oviduct estrogen receptor had the samemore » characteristics as those in other estrogen target tissues and was present in the same amount as in adult female Xenopus hepatocytes. The successful establishment and characterization of primary cultures of both liver and oviduct cells now fulfill the conditions required for investigating the basis for tissue specificity of regulation by estrogen of Xenopus egg protein gene expression in primary cell culture.« less

ATP hydrolysis in Eg5 kinesin involves a catalytic two-water mechanism.

PubMed

Parke, Courtney L; Wojcik, Edward J; Kim, Sunyoung; Worthylake, David K

2010-02-19

Motor proteins couple steps in ATP binding and hydrolysis to conformational switching both in and remote from the active site. In our kinesin.AMPPPNP crystal structure, closure of the active site results in structural transformations appropriate for microtubule binding and organizes an orthosteric two-water cluster. We conclude that a proton is shared between the lytic water, positioned for gamma-phosphate attack, and a second water that serves as a general base. To our knowledge, this is the first experimental detection of the catalytic base for any ATPase. Deprotonation of the second water by switch residues likely triggers subsequent large scale structural rearrangements. Therefore, the catalytic base is responsible for initiating nucleophilic attack of ATP and for relaying the positive charge over long distances to initiate mechanotransduction. Coordination of switch movements via sequential proton transfer along paired water clusters may be universal for nucleotide triphosphatases with conserved active sites, such as myosins and G-proteins.

Pressure-Induced Changes in the Structure and Function of the Kinesin-Microtubule Complex

PubMed Central

Nishiyama, Masayoshi; Kimura, Yoshifumi; Nishiyama, Yoshio; Terazima, Masahide

2009-01-01

Kinesin-1 is an ATP-driven molecular motor that “walks” along a microtubule by working two heads in a “hand-over-hand” fashion. The stepping motion is well-coordinated by intermolecular interactions between the kinesin head and microtubule, and is sensitively changed by applied forces. We demonstrate that hydrostatic pressure works as an inhibitory action on kinesin motility. We developed a high-pressure microscope that enables the application of hydrostatic pressures of up to 200 MPa (2000 bar). Under high-pressure conditions, taxol-stabilized microtubules were shortened from both ends at the same speed. The sliding velocity of kinesin motors was reversibly changed by pressure, and reached half-maximal value at ∼100 MPa. The pressure-velocity relationship was very close to the force-velocity relationship of single kinesin molecules, suggesting a similar inhibitory mechanism on kinesin motility. Further analysis showed that the pressure mainly affects the stepping motion, but not the ATP binding reaction. The application of pressure is thought to enhance the structural fluctuation and/or association of water molecules with the exposed regions of the kinesin head and microtubule. These pressure-induced effects could prevent kinesin motors from completing the stepping motion. PMID:19186149

Mitotic Kinesin CENP-E Promotes Microtubule Plus-End Elongation

PubMed Central

Sardar, Harjinder S.; Luczak, Vincent G.; Lopez, Maria M.; Lister, Bradford C.; Gilbert, Susan P.

2010-01-01

Summary Centromere protein CENP-E is a dimeric kinesin (Kinesin-7 family) with critical roles in mitosis including establishment of microtubule (MT)-chromosome linkage and movement of monooriented chromosomes on kinetochore microtubules (kMTs) for proper alignment at metaphase [1-9]. We performed studies to test the hypothesis that CENP-E promotes MT elongation at the MT plus-ends. A human CENP-E construct was engineered, expressed, and purified which yielded the CENP-E-6His dimeric motor protein. The results show that CENP-E promotes MT plus-end directed MT gliding at 11 nm/s. The results from real-time microscopy assays indicate that 60.3% of polarity marked MTs exhibited CENP-E promoted MT plus-end elongation. The MT extension required ATP turnover, and MT plus-end elongation occurred at 1.48 μm/30 min. Immunolocalization studies revealed that 80.8% of plus-end elongated MTs showed CENP-E at the MT plus-end. The time dependence of CENP-E promoted MT elongation in solution best fit a single exponential function (kobs = 5.1 s−1), which is indicative of a mechanism in which α,β-tubulin subunit addition is tightly coupled to ATP turnover. Based on these results, we propose that CENP-E as part of its function in chromosome kinetochore-MT linkage plays a direct role in MT elongation. PMID:20797864

Tug-of-war of microtubule filaments at the boundary of a kinesin- and dynein-patterned surface

NASA Astrophysics Data System (ADS)

Ikuta, Junya; Kamisetty, Nagendra K.; Shintaku, Hirofumi; Kotera, Hidetoshi; Kon, Takahide; Yokokawa, Ryuji

2014-06-01

Intracellular cargo is transported by multiple motor proteins. Because of the force balance of motors with mixed polarities, cargo moves bidirectionally to achieve biological functions. Here, we propose a microtubule gliding assay for a tug-of-war study of kinesin and dynein. A boundary of the two motor groups is created by photolithographically patterning gold to selectively attach kinesin to the glass and dynein to the gold surface using a self-assembled monolayer. The relationship between the ratio of two antagonistic motor numbers and the velocity is derived from a force-velocity relationship for each motor to calculate the detachment force and motor backward velocity. Although the tug-of-war involves >100 motors, values are calculated for a single molecule and reflect the collective dynein and non-collective kinesin functions when they work as a team. This assay would be useful for detailed in vitro analysis of intracellular motility, e.g., mitosis, where a large number of motors with mixed polarities are involved.

Tug-of-war of microtubule filaments at the boundary of a kinesin- and dynein-patterned surface

PubMed Central

Ikuta, Junya; Kamisetty, Nagendra K.; Shintaku, Hirofumi; Kotera, Hidetoshi; Kon, Takahide; Yokokawa, Ryuji

2014-01-01

Intracellular cargo is transported by multiple motor proteins. Because of the force balance of motors with mixed polarities, cargo moves bidirectionally to achieve biological functions. Here, we propose a microtubule gliding assay for a tug-of-war study of kinesin and dynein. A boundary of the two motor groups is created by photolithographically patterning gold to selectively attach kinesin to the glass and dynein to the gold surface using a self-assembled monolayer. The relationship between the ratio of two antagonistic motor numbers and the velocity is derived from a force-velocity relationship for each motor to calculate the detachment force and motor backward velocity. Although the tug-of-war involves >100 motors, values are calculated for a single molecule and reflect the collective dynein and non-collective kinesin functions when they work as a team. This assay would be useful for detailed in vitro analysis of intracellular motility, e.g., mitosis, where a large number of motors with mixed polarities are involved. PMID:24923426

Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell-mediated cytotoxicity.

PubMed

Tuli, Amit; Thiery, Jerome; James, Ashley M; Michelet, Xavier; Sharma, Mahak; Garg, Salil; Sanborn, Keri B; Orange, Jordan S; Lieberman, Judy; Brenner, Michael B

2013-12-01

Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell-mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity.

Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell–mediated cytotoxicity

PubMed Central

Tuli, Amit; Thiery, Jerome; James, Ashley M.; Michelet, Xavier; Sharma, Mahak; Garg, Salil; Sanborn, Keri B.; Orange, Jordan S.; Lieberman, Judy; Brenner, Michael B.

2013-01-01

Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell–mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity. PMID:24088571

Drosophila Shaking-B protein forms gap junctions in paired Xenopus oocytes.

PubMed

Phelan, P; Stebbings, L A; Baines, R A; Bacon, J P; Davies, J A; Ford, C

1998-01-08

In most multicellular organisms direct cell-cell communication is mediated by the intercellular channels of gap junctions. These channels allow the exchange of ions and molecules that are believed to be essential for cell signalling during development and in some differentiated tissues. Proteins called connexins, which are products of a multigene family, are the structural components of vertebrate gap junctions. Surprisingly, molecular homologues of the connexins have not been described in any invertebrate. A separate gene family, which includes the Drosophila genes shaking-B and l(1)ogre, and the Caenorhabditis elegans genes unc-7 and eat-5, encodes transmembrane proteins with a predicted structure similar to that of the connexins. shaking-B and eat-5 are required for the formation of functional gap junctions. To test directly whether Shaking-B is a channel protein, we expressed it in paired Xenopus oocytes. Here we show that Shaking-B localizes to the membrane, and that its presence induces the formation of functional intercellular channels. To our knowledge, this is the first structural component of an invertebrate gap junction to be characterized.

Single molecule FRET observation of kinesin-1’s head-tail interaction on microtubule

PubMed Central

Aoki, Takahiro; Tomishige, Michio; Ariga, Takayuki

2013-01-01

Kinesin-1 (conventional kinesin) is a molecular motor that transports various cargo such as endoplasmic reticulum and mitochondria in cells. Its two head domains walk along microtubule by hydrolyzing ATP, while the tail domains at the end of the long stalk bind to the cargo. When a kinesin is not carrying cargo, its motility and ATPase activity is inhibited by direct interactions between the tail and head. However, the mechanism of this tail regulation is not well understood. Here, we apply single molecule fluorescence resonance energy transfer (smFRET) to observe this interaction in stalk-truncated kinesin. We found that kinesin with two tails forms a folding conformation and dissociates from microtubules, whereas kinesin with one tail remains bound to the micro-tubule and is immobile even in the presence of ATP. We further investigated the head-tail interaction as well as head-head coordination on the microtubule at various nucleotide conditions. From these results, we propose a two-step inhibition model for kinesin motility. PMID:27493553

Xenopus LAP2β protein knockdown affects location of lamin B and nucleoporins and has effect on assembly of cell nucleus and cell viability.

PubMed

Dubińska-Magiera, Magda; Chmielewska, Magdalena; Kozioł, Katarzyna; Machowska, Magdalena; Hutchison, Christopher J; Goldberg, Martin W; Rzepecki, Ryszard

2016-05-01

Xenopus LAP2β protein is the single isoform expressed in XTC cells. The protein localizes on heterochromatin clusters both at the nuclear envelope and inside a cell nucleus. The majority of XLAP2β fraction neither colocalizes with TPX2 protein during interphase nor can be immunoprecipitated with XLAP2β antibody. Knockdown of the XLAP2β protein expression in XTC cells by synthetic siRNA and plasmid encoded siRNA resulted in nuclear abnormalities including changes in shape of nuclei, abnormal chromatin structure, loss of nuclear envelope, mislocalization of integral membrane proteins of INM such as lamin B2, mislocalization of nucleoporins, and cell death. Based on timing of cell death, we suggest mechanism associated with nucleus reassembly or with entry into mitosis. This confirms that Xenopus LAP2 protein is essential for the maintenance of cell nucleus integrity and the process of its reassembly after mitosis.

Amyloid-like Self-Assembly of a Cellular Compartment.

PubMed

Boke, Elvan; Ruer, Martine; Wühr, Martin; Coughlin, Margaret; Lemaitre, Regis; Gygi, Steven P; Alberti, Simon; Drechsel, David; Hyman, Anthony A; Mitchison, Timothy J

2016-07-28

Most vertebrate oocytes contain a Balbiani body, a large, non-membrane-bound compartment packed with RNA, mitochondria, and other organelles. Little is known about this compartment, though it specifies germline identity in many non-mammalian vertebrates. We show Xvelo, a disordered protein with an N-terminal prion-like domain, is an abundant constituent of Xenopus Balbiani bodies. Disruption of the prion-like domain of Xvelo, or substitution with a prion-like domain from an unrelated protein, interferes with its incorporation into Balbiani bodies in vivo. Recombinant Xvelo forms amyloid-like networks in vitro. Amyloid-like assemblies of Xvelo recruit both RNA and mitochondria in binding assays. We propose that Xenopus Balbiani bodies form by amyloid-like assembly of Xvelo, accompanied by co-recruitment of mitochondria and RNA. Prion-like domains are found in germ plasm organizing proteins in other species, suggesting that Balbiani body formation by amyloid-like assembly could be a conserved mechanism that helps oocytes function as long-lived germ cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Human neuronal stargazin-like proteins, γ2, γ3 and γ4; an investigation of their specific localization in human brain and their influence on CaV2.1 voltage-dependent calcium channels expressed in Xenopus oocytes.

PubMed Central

Moss, Fraser J; Dolphin, Annette C; Clare, Jeffrey J

2003-01-01

Background Stargazin (γ2) and the closely related γ3, and γ4 transmembrane proteins are part of a family of proteins that may act as both neuronal voltage-dependent calcium channel (VDCC) γ subunits and transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazoleproponinc (AMPA) receptor regulatory proteins (TARPs). In this investigation, we examined the distribution patterns of the stargazin-like proteins γ2, γ3, and γ4 in the human central nervous system (CNS). In addition, we investigated whether human γ2 or γ4 could modulate the electrophysiological properties of a neuronal VDCC complex transiently expressed in Xenopus oocytes. Results The mRNA encoding human γ2 is highly expressed in cerebellum, cerebral cortex, hippocampus and thalamus, whereas γ3 is abundant in cerebral cortex and amygdala and γ4 in the basal ganglia. Immunohistochemical analysis of the cerebellum determined that both γ2 and γ4 are present in the molecular layer, particularly in Purkinje cell bodies and dendrites, but have an inverse expression pattern to one another in the dentate cerebellar nucleus. They are also detected in the interneurons of the granule cell layer though only γ2 is clearly detected in granule cells. The hippocampus stains for γ2 and γ4 throughout the layers of the every CA region and the dentate gyrus, whilst γ3 appears to be localized particularly to the pyramidal and granule cell bodies. When co-expressed in Xenopus oocytes with a CaV2.1/β4 VDCC complex, either in the absence or presence of an α2δ2 subunit, neither γ2 nor γ4 significantly modulated the VDCC peak current amplitude, voltage-dependence of activation or voltage-dependence of steady-state inactivation. Conclusion The human γ2, γ3 and γ4 stargazin-like proteins are detected only in the CNS and display differential distributions among brain regions and several cell types in found in the cerebellum and hippocampus. These distribution patterns closely resemble those reported by other

Metallic Glass Wire Based Localization of Kinesin/Microtubule Bio-molecular Motility System

NASA Astrophysics Data System (ADS)

Kim, K.; Sikora, A.; Yaginuma, S.; Nakayama, K. S.; Nakazawa, H.; Umetsu, M.; Hwang, W.; Teizer, W.

2014-03-01

We report electrophoretic accumulation of microtubules along metallic glass (Pd42.5Cu30Ni7.5P20) wires free-standing in solution. Microtubules are dynamic cytoskeletal filaments. Kinesin is a cytoskeletal motor protein. Functions of these bio-molecules are central to various dynamic cellular processes. Functional artificial organization of bio-molecules is a prerequisite for transferring their native functions into device applications. Fluorescence microscopy at the individual-microtubule level reveals microtubules aligning along the wire axis during the electrophoretic migration. Casein-treated electrodes are effective for releasing trapped microtubules upon removal of the external field. Furthermore, we demonstrate gliding motion of microtubules on kinesin-treated metallic glass wires. The reversible manner in the local adsorption of microtubules, the flexibility of wire electrodes, and the compatibility between the wire electrode and the bio-molecules are beneficial for spatio-temporal manipulation of the motility machinery in 3 dimensions.

Functional diversification of the kinesin-14 family in land plants.

PubMed

Gicking, Allison M; Swentowsky, Kyle W; Dawe, R Kelly; Qiu, Weihong

2018-05-12

In most eukaryotes, cytoplasmic dynein serves as the primary cytoskeletal motor for minus-end-directed processes along microtubules. However, land plants lack dynein, having instead a large number of kinesin-14s, which suggests that kinesin-14s may have evolved to fill the cellular niche left by dynein. In addition, land plants do not have centrosomes, but contain specialized microtubule-based structures called phragmoplasts that facilitate the formation of new cell walls following cell division. This Review aims to compile the evidence for functional diversification of kinesin-14s in land plants. Known functions include spindle morphogenesis, microtubule-based trafficking, nuclear migration, chloroplast distribution, and phragmoplast expansion. Plant kinesin-14s have also evolved direct roles in chromosome segregation in maize and novel biochemical features such as actin transport and processive motility in the homodimeric state. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Molecular properties of the N-terminal extension of the fission yeast kinesin-5, Cut7.

PubMed

Edamatsu, M

2016-02-11

Kinesin-5 plays an essential role in spindle formation and function, and serves as a potential target for anti-cancer drugs. The aim of this study was to elucidate the molecular properties of the N-terminal extension of the Schizosaccharomyces pombe kinesin-5, Cut7. This extension is rich in charged amino acids and predicted to be intrinsically disordered. In S. pombe cells, a Cut7 construct lacking half the N-terminal extension failed to localize along the spindle microtubules and formed a monopolar spindle. However, a construct lacking the entire N-terminal extension exhibited normal localization and formed a typical bipolar spindle. In addition, in vitro analyses revealed that the truncated Cut7 constructs demonstrated similar motile velocities and directionalities as the wild-type motor protein, but the microtubule landing rates were significantly reduced. These findings suggest that the N-terminal extension is not required for normal Cut7 intracellular localization or function, but alters the microtubule-binding properties of this protein in vitro.

Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity

PubMed Central

Grover, Rahul; Fischer, Janine; Schwarz, Friedrich W.; Walter, Wilhelm J.; Schwille, Petra; Diez, Stefan

2016-01-01

In eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to nonbiological substrates. However, the collective transport by membrane-anchored motors, that is, motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors’ anchorage to a lipid bilayer on the collective transport characteristics. We reconstituted “membrane-anchored” gliding motility assays using truncated kinesin-1 motors with a streptavidin-binding peptide tag that can attach to streptavidin-loaded, supported lipid bilayers. We found that the diffusing kinesin-1 motors propelled the microtubules in the presence of ATP. Notably, we found the gliding velocity of the microtubules to be strongly dependent on the number of motors and their diffusivity in the lipid bilayer. The microtubule gliding velocity increased with increasing motor density and membrane viscosity, reaching up to the stepping velocity of single motors. This finding is in contrast to conventional gliding motility assays where the density of surface-immobilized kinesin-1 motors does not influence the microtubule velocity over a wide range. We reason that the transport efficiency of membrane-anchored motors is reduced because of their slippage in the lipid bilayer, an effect that we directly observed using single-molecule fluorescence microscopy. Our results illustrate the importance of motor–cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport. PMID:27803325

Xenopus laevis ribosomal protein genes: isolation of recombinant cDNA clones and study of the genomic organization.

PubMed Central

Bozzoni, I; Beccari, E; Luo, Z X; Amaldi, F

1981-01-01

Poly-A+ mRNA from Xenopus laevis oocytes, partially enriched for r-protein coding capacity has been used as starting material for preparing a cDNA bank in plasmid pBR322. The clones containing sequences specific for r-proteins have been selected by translation of the complementary mRNAs. Clones for six different r-proteins have been identified and utilized as probes for studying their genomic organization. Two gene copies per haploid genome were found for r-proteins L1, L14, S19, and four-five for protein S1, S8 and L32. Moreover a population polymorphism has been observed for the genomic regions containing sequences for r-protein S1, S8 and L14. Images PMID:6112733

Tau excess impairs mitosis and kinesin-5 function, leading to aneuploidy and cell death.

PubMed

Bougé, Anne-Laure; Parmentier, Marie-Laure

2016-03-01

In neurodegenerative diseases such as Alzheimer's disease (AD), cell cycle defects and associated aneuploidy have been described. However, the importance of these defects in the physiopathology of AD and the underlying mechanistic processes are largely unknown, in particular with respect to the microtubule (MT)-binding protein Tau, which is found in excess in the brain and cerebrospinal fluid of affected individuals. Although it has long been known that Tau is phosphorylated during mitosis to generate a lower affinity for MTs, there is, to our knowledge, no indication that an excess of this protein could affect mitosis. Here, we studied the effect of an excess of human Tau (hTau) protein on cell mitosis in vivo. Using the Drosophila developing wing disc epithelium as a model, we show that an excess of hTau induces a mitotic arrest, with the presence of monopolar spindles. This mitotic defect leads to aneuploidy and apoptotic cell death. We studied the mechanism of action of hTau and found that the MT-binding domain of hTau is responsible for these defects. We also demonstrate that the effects of hTau occur via the inhibition of the function of the kinesin Klp61F, the Drosophila homologue of kinesin-5 (also called Eg5 or KIF11). We finally show that this deleterious effect of hTau is also found in other Drosophila cell types (neuroblasts) and tissues (the developing eye disc), as well as in human HeLa cells. By demonstrating that MT-bound Tau inhibits the Eg5 kinesin and cell mitosis, our work provides a new framework to consider the role of Tau in neurodegenerative diseases. © 2016. Published by The Company of Biologists Ltd.

Metabolism of ribosomal proteins microinjected into the oocytes of Xenopus laevis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsurugi, K.; Motizuki, M.; Mitsui, K.

1988-01-01

When the total proteins from Xenopus laevis 60 S ribosomal subunits (TP60) were /sup 3/H-labeled in vitro and injected back into X. laevis oocytes, most /sup 3/H-TP60 are integrated into the cytoplasmic 60 S subunits via the nucleus during 16 h of incubation. In the oocytes whose rRNA synthesis is inhibited, /sup 3/H-TP60 are rapidly degraded with a half-life of 2-3 h. This degradation ceased as soon as rRNA synthesis was resumed, suggesting that ribosomal proteins unassociated with nascent rRNA are unstable in the oocytes. The degradation of /sup 3/H-TP60 in the absence of RNA synthesis was inhibited by iodoacetamide,more » a cysteine protease inhibitor, resulting in the accumulation of /sup 3/H-TP60 in the nucleus reaching about a threefold concentration in the cytoplasm. Considering the results with enucleated oocytes, we suggest that the X. laevis nucleus has a limited capacity to accumulate ribosomal proteins in an active manner but that those ribosomal proteins accumulated in excess over rRNA synthesis are degraded by a cysteine protease in the nucleus. By contrast, ribosomal proteins from Escherichia coli only equilibrate between the nucleus and the cytoplasm and are degraded by serine protease(s) in the cytoplasm without being integrated in the form of ribosomes in the nucleus.« less

Restraint of presynaptic protein levels by Wnd/DLK signaling mediates synaptic defects associated with the kinesin-3 motor Unc-104

PubMed Central

Asghari Adib, Elham; Stanchev, Doychin T; Xiong, Xin; Klinedinst, Susan; Soppina, Pushpanjali; Jahn, Thomas Robert; Hume, Richard I

2017-01-01

The kinesin-3 family member Unc-104/KIF1A is required for axonal transport of many presynaptic components to synapses, and mutation of this gene results in synaptic dysfunction in mice, flies and worms. Our studies at the Drosophila neuromuscular junction indicate that many synaptic defects in unc-104-null mutants are mediated independently of Unc-104’s transport function, via the Wallenda (Wnd)/DLK MAP kinase axonal damage signaling pathway. Wnd signaling becomes activated when Unc-104’s function is disrupted, and leads to impairment of synaptic structure and function by restraining the expression level of active zone (AZ) and synaptic vesicle (SV) components. This action concomitantly suppresses the buildup of synaptic proteins in neuronal cell bodies, hence may play an adaptive role to stresses that impair axonal transport. Wnd signaling also becomes activated when pre-synaptic proteins are over-expressed, suggesting the existence of a feedback circuit to match synaptic protein levels to the transport capacity of the axon. PMID:28925357

Disruption of Axonal Transport Perturbs Bone Morphogenetic Protein (BMP) - Signaling and Contributes to Synaptic Abnormalities in Two Neurodegenerative Diseases

PubMed Central

Kang, Min Jung; Hansen, Timothy J.; Mickiewicz, Monique; Kaczynski, Tadeusz J.; Fye, Samantha; Gunawardena, Shermali

2014-01-01

Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. Here we show that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). We found that components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility was also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also showed synaptic morphological defects. Strikingly TKV motility and p-Mad signaling were disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP) with a familial Alzheimer's disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins showed accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together our results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exist. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases. PMID:25127478

Temporal control of bidirectional lipid-droplet motion in Drosophila depends on the ratio of kinesin-1 and its co-factor Halo

PubMed Central

Arora, Gurpreet K.; Tran, Susan L.; Rizzo, Nicholas; Jain, Ankit; Welte, Michael A.

2016-01-01

ABSTRACT During bidirectional transport, individual cargoes move continuously back and forth along microtubule tracks, yet the cargo population overall displays directed net transport. How such transport is controlled temporally is not well understood. We analyzed this issue for bidirectionally moving lipid droplets in Drosophila embryos, a system in which net transport direction is developmentally controlled. By quantifying how the droplet distribution changes as embryos develop, we characterize temporal transitions in net droplet transport and identify the crucial contribution of the previously identified, but poorly characterized, transacting regulator Halo. In particular, we find that Halo is transiently expressed; rising and falling Halo levels control the switches in global distribution. Rising Halo levels have to pass a threshold before net plus-end transport is initiated. This threshold level depends on the amount of the motor kinesin-1: the more kinesin-1 is present, the more Halo is needed before net plus-end transport commences. Because Halo and kinesin-1 are present in common protein complexes, we propose that Halo acts as a rate-limiting co-factor of kinesin-1. PMID:26906417

The Nedd4 binding protein 3 is required for anterior neural development in Xenopus laevis.

PubMed

Kiem, Lena-Maria; Dietmann, Petra; Linnemann, Alexander; Schmeisser, Michael J; Kühl, Susanne J

2017-03-01

The Fezzin family member Nedd4-binding protein 3 (N4BP3) is known to regulate axonal and dendritic branching. Here, we show that n4bp3 is expressed in the neural tissue of the early Xenopus laevis embryo including the eye, the brain and neural crest cells. Knockdown of N4bp3 in the Xenopus anterior neural tissue results in severe developmental impairment of the eye, the brain and neural crest derived cranial cartilage structures. Moreover, we demonstrate that N4bp3 depletion leads to a significant reduction of both eye and brain specific marker genes and reduced neural crest cell migration. Finally, we demonstrate an impact of N4bp3 deficiency on cell apoptosis and proliferation. Our studies indicate that N4bp3 is required for early anterior neural development of vertebrates. This is in line with a study implicating that genetic disruption of N4BP3 in humans might be related to neurodevelopmental disease. Copyright © 2017 Elsevier Inc. All rights reserved.

Nucleotide-induced conformations in the neck region of dimeric kinesin

PubMed Central

Skiniotis, Georgios; Surrey, Thomas; Altmann, Stephan; Gross, Heinz; Song, Young-Hwa; Mandelkow, Eckhard; Hoenger, Andreas

2003-01-01

The neck region of kinesin constitutes a key component in the enzyme’s walking mechanism. Here we applied cryoelectron microscopy and image reconstruction to investigate the location of the kinesin neck in dimeric and monomeric constructs complexed to microtubules. To this end we enhanced the visibility of this region by engineering an SH3 domain into the transition between neck linker and neck coiled coil. The resulting chimeric kinesin constructs remained functional as verified by physiology assays. In the presence of AMP–PNP the SH3 domains allowed us to identify the position of the neck in a well defined conformation and revealed its high flexibility in the absence of nucleotide. We show here the double-headed binding of dimeric kinesin along the same protofilament, which is characterized by the opposite directionality of neck linkers. In this configuration the neck coiled coil appears fully zipped. The position of the neck region in dimeric constructs is not affected by the presence of the tubulin C-termini as confirmed by subtilisin treatment of microtubules prior to motor decoration. PMID:12660159

Expression patterns of Xenopus FGF receptor-like 1/nou-darake in early Xenopus development resemble those of planarian nou-darake and Xenopus FGF8.

PubMed

Hayashi, Shuichi; Itoh, Mari; Taira, Sumiko; Agata, Kiyokazu; Taira, Masanori

2004-08-01

Fibroblast growth factors (FGFs) mediate many cell-to-cell signaling events during early development. Nou-darake (ndk), a gene encoding an FGF receptor (FGFR)-like molecule, was found to be highly and specifically expressed in the head region of the planarian Dugesia japonica, and its functional analyses provided strong molecular evidence for the existence of a brain-inducing circuit based on the FGF signaling pathway. To analyze the role of ndk during vertebrate development, we isolated the Xenopus ortholog of ndk, the vertebrate FGFR-like 1 gene (XFGFRL1). Expression of XFGFRL1/Xndk was first detected in the anterior region at the late gastrula stage and dramatically increased at the early neurula stage in an overall anterior mesendodermal region, including the prechordal plate, paraxial mesoderm, anterior endoderm, and archenteron roof. This anterior expression pattern resembles that of ndk in planarians, suggesting that the expression of FGFRL1/ndk is conserved in evolution between these two distantly diverged organisms. During the tail bud stages, XFGFRL1/Xndk expression was detected in multiple regions, including the forebrain, eyes, midbrain-hindbrain boundary, otic vesicles, visceral arches, and somites. In many of these regions, XFGFRL1/Xndk was coexpressed with XFGF8, indicating that XFGFRL1/Xndk is a member of the XFGF8 synexpression group, which includes sprouty, sef, and isthmin. Copyright 2004 Wiley-Liss, Inc.

Delivery of kinesin spindle protein targeting siRNA in solid lipid nanoparticles to cellular models of tumor vasculature

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ying, Bo; Campbell, Robert B., E-mail: robert.campbell@mcphs.edu

2014-04-04

Highlights: • siRNA-lipid nanoparticles are solid particles not lipid bilayers with aqueous core. • High, but not low, PEG content can prevent nanoparticle encapsulation of siRNA. • PEG reduces cellular toxicity of cationic nanoparticles in vitro. • PEG reduces zeta potential while improving gene silencing of siRNA nanoparticles. • Kinesin spindle protein can be an effective target for tumor vascular targeting. - Abstract: The ideal siRNA delivery system should selectively deliver the construct to the target cell, avoid enzymatic degradation, and evade uptake by phagocytes. In the present study, we evaluated the importance of polyethylene glycol (PEG) on lipid-based carriermore » systems for encapsulating, and delivering, siRNA to tumor vessels using cellular models. Lipid nanoparticles containing different percentage of PEG were evaluated based on their physical chemical properties, density compared to water, siRNA encapsulation, toxicity, targeting efficiency and gene silencing in vitro. siRNA can be efficiently loaded into lipid nanoparticles (LNPs) when DOTAP is included in the formulation mixture. However, the total amount encapsulated decreased with increase in PEG content. In the presence of siRNA, the final formulations contained a mixed population of particles based on density. The major population which contains the majority of siRNA exhibited a density of 4% glucose, and the minor fraction associated with a decreased amount of siRNA had a density less than PBS. The inclusion of 10 mol% PEG resulted in a greater amount of siRNA associated with the minor fraction. Finally, when kinesin spindle protein (KSP) siRNA was encapsulated in lipid nanoparticles containing a modest amount of PEG, the proliferation of endothelial cells was inhibited due to the efficient knock down of KSP mRNA. The presence of siRNA resulted in the formation of solid lipid nanoparticles when prepared using the thin film and hydration method. LNPs with a relatively modest

Xenopus cytoplasmic linker–associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules

PubMed Central

Marx, Astrid; Godinez, William J.; Tsimashchuk, Vasil; Bankhead, Peter; Rohr, Karl; Engel, Ulrike

2013-01-01

Dynamic microtubules (MTs) are required for neuronal guidance, in which axons extend directionally toward their target tissues. We found that depletion of the MT-binding protein Xenopus cytoplasmic linker–associated protein 1 (XCLASP1) or treatment with the MT drug Taxol reduced axon outgrowth in spinal cord neurons. To quantify the dynamic distribution of MTs in axons, we developed an automated algorithm to detect and track MT plus ends that have been fluorescently labeled by end-binding protein 3 (EB3). XCLASP1 depletion reduced MT advance rates in neuronal growth cones, very much like treatment with Taxol, demonstrating a potential link between MT dynamics in the growth cone and axon extension. Automatic tracking of EB3 comets in different compartments revealed that MTs increasingly slowed as they passed from the axon shaft into the growth cone and filopodia. We used speckle microscopy to demonstrate that MTs experience retrograde flow at the leading edge. Microtubule advance in growth cone and filopodia was strongly reduced in XCLASP1-depleted axons as compared with control axons, but actin retrograde flow remained unchanged. Instead, we found that XCLASP1-depleted growth cones lacked lamellipodial actin organization characteristic of protrusion. Lamellipodial architecture depended on XCLASP1 and its capacity to associate with MTs, highlighting the importance of XCLASP1 in actin–microtubule interactions. PMID:23515224

Structure-based molecular simulations reveal the enhancement of biased Brownian motions in single-headed kinesin.

PubMed

Kanada, Ryo; Kuwata, Takeshi; Kenzaki, Hiroo; Takada, Shoji

2013-01-01

Kinesin is a family of molecular motors that move unidirectionally along microtubules (MT) using ATP hydrolysis free energy. In the family, the conventional two-headed kinesin was experimentally characterized to move unidirectionally through "walking" in a hand-over-hand fashion by coordinated motions of the two heads. Interestingly a single-headed kinesin, a truncated KIF1A, still can generate a biased Brownian movement along MT, as observed by in vitro single molecule experiments. Thus, KIF1A must use a different mechanism from the conventional kinesin to achieve the unidirectional motions. Based on the energy landscape view of proteins, for the first time, we conducted a set of molecular simulations of the truncated KIF1A movements over an ATP hydrolysis cycle and found a mechanism exhibiting and enhancing stochastic forward-biased movements in a similar way to those in experiments. First, simulating stand-alone KIF1A, we did not find any biased movements, while we found that KIF1A with a large friction cargo-analog attached to the C-terminus can generate clearly biased Brownian movements upon an ATP hydrolysis cycle. The linked cargo-analog enhanced the detachment of the KIF1A from MT. Once detached, diffusion of the KIF1A head was restricted around the large cargo which was located in front of the head at the time of detachment, thus generating a forward bias of the diffusion. The cargo plays the role of a diffusional anchor, or cane, in KIF1A "walking."

Construction of artificial cilia from microtubules and kinesins through a well-designed bottom-up approach.

PubMed

Sasaki, Ren; Kabir, Arif Md Rashedul; Inoue, Daisuke; Anan, Shizuka; Kimura, Atsushi P; Konagaya, Akihiko; Sada, Kazuki; Kakugo, Akira

2018-04-05

Self-organized structures of biomolecular motor systems, such as cilia and flagella, play key roles in the dynamic processes of living organisms, like locomotion or the transportation of materials. Although fabrication of such self-organized structures from reconstructed biomolecular motor systems has attracted much attention in recent years, a systematic construction methodology is still lacking. In this work, through a bottom-up approach, we fabricated artificial cilia from a reconstructed biomolecular motor system, microtubule/kinesin. The artificial cilia exhibited a beating motion upon the consumption, by the kinesins, of the chemical energy obtained from the hydrolysis of adenosine triphosphate (ATP). Several design parameters, such as the length of the microtubules, the density of the kinesins along the microtubules, the depletion force among the microtubules, etc., have been identified, which permit tuning of the beating frequency of the artificial cilia. The beating frequency of the artificial cilia increases upon increasing the length of the microtubules, but declines for the much longer microtubules. A high density of the kinesins along the microtubules is favorable for the beating motion of the cilia. The depletion force induced bundling of the microtubules accelerated the beating motion of the artificial cilia and increased the beating frequency. This work helps understand the role of self-assembled structures of the biomolecular motor systems in the dynamics of living organisms and is expected to expedite the development of artificial nanomachines, in which the biomolecular motors may serve as actuators.

Isolation and characterization of Xenopus laevis homologs of the mouse inv gene and functional analysis of the conserved calmodulin binding sites.

PubMed

Yasuhiko, Yukuto; Shiokawa, Koichiro; Mochizuki, Toshio; Asashima, Makoto; Yokoyama, Takahiko

2006-04-01

The homozygous inv (inversion of embryonic turning) mouse mutant shows situs inversus and polycystic kidney disease, both of which result from the lack of the inv gene. Previously, we suggested that inv may be important for the left-right axis formation, not only in mice but also in Xenopus, and that calmodulin regulates this inv protein function. Here, we isolated and characterized two Xenopus laevis homologs (Xinv-1 and Xinv-2) of the mouse inv gene, and performed functional analysis of the conserved IQ motifs that interact with calmodulin. Xinv-1 expresses early in development in the same manner as mouse inv does. Unexpectedly, a full-length Xenopus inv mRNA did not randomize cardiac orientation when injected into Xenopus embryos, which is different from mouse inv mRNA. Contrary to mouse inv mRNA, Xenopus inv mRNA with mutated IQ randomized cardiac orientation. The present study indicates that calmodulin binding sites (IQ motifs) are crucial in controlling the biological activity of both mouse and Xenopus inv proteins. Although mouse and Xenopus inv genes have a quite similar structure, the interaction with calmodulin and IQ motifs of Xenopus inv and mouse inv proteins may regulate their function in different ways.

Bidirectional motility of the fission yeast kinesin-5, Cut7

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edamatsu, Masaki, E-mail: cedam@mail.ecc.u-tokyo.ac.jp

Highlights: • Motile properties of Cut7 (fission yeast kinesin-5) were studied for the first time. • Half-length Cut7 moved toward plus-end direction of microtubule. • Full-length Cut7 moved toward minus-end direction of microtubule. • N- and C-terminal microtubule binding sites did not switch the motile direction. - Abstract: Kinesin-5 is a homotetrameric motor with its motor domain at the N-terminus. Kinesin-5 crosslinks microtubules and functions in separating spindle poles during mitosis. In this study, the motile properties of Cut7, fission yeast kinesin-5, were examined for the first time. In in vitro motility assays, full-length Cut7 moved toward minus-end of microtubules,more » but the N-terminal half of Cut7 moved toward the opposite direction. Furthermore, additional truncated constructs lacking the N-terminal or C-terminal regions, but still contained the motor domain, did not switch the motile direction. These indicated that Cut7 was a bidirectional motor, and microtubule binding regions at the N-terminus and C-terminus were not involved in its directionality.« less

A Unique Role for Endothelial Cell Kinesin Light Chain 1, Variant 1 in Leukocyte Transendothelial Migration

PubMed Central

Cyrus, Bita F.; Muller, William A.

2017-01-01

A reservoir of parajunctional membrane in endothelial cells, the lateral border recycling compartment (LBRC), is critical for transendothelial migration (TEM). We have previously shown that targeted recycling of the LBRC to the site of TEM requires microtubules and a kinesin molecular motor. However, the identity of the kinesin and mechanism of cargo binding were not known. We show that microinjection of endothelial cells with a monoclonal antibody specific for kinesin-1 significantly blocked LBRC-targeted recycling and TEM. In complementary experiments, knocking down KIF5B, a ubiquitous kinesin-1 isoform, in endothelial cells significantly decreased targeted recycling of the LBRC and leukocyte TEM. Kinesin heavy chains move cargo along microtubules by one of many kinesin light chains (KLCs), which directly bind the cargo. Knocking down KLC 1 isoform variant 1 (KLC1C) significantly decreased LBRC-targeted recycling and TEM, whereas knocking down other isoforms of KLC1 had no effect. Re-expression of KLC1C resistant to the knockdown shRNA restored targeted recycling and TEM. Thus kinesin-1 and KLC1C are specifically required for targeted recycling and TEM. These data suggest that of the many potential combinations of the 45 kinesin family members and multiple associated light chains, KLC1C links the LBRC to kinesin-1 (KIF5B) during targeted recycling and TEM. Thus, KLC1C can potentially be used as a target for anti-inflammatory therapy. PMID:26994343

A Homolog of Subtilisin-Like Proprotein Convertase 7 Is Essential to Anterior Neural Development in Xenopus

PubMed Central

Senturker, Sema; Thomas, John Terrig; Mateshaytis, Jennifer; Moos, Malcolm

2012-01-01

Background Subtilisin-like Proprotein Convertase 7 (SPC7) is a member of the subtilisin/kexin family of pro-protein convertases. It cleaves many pro-proteins to release their active proteins, including members of the bone morphogenetic protein (BMP) family of signaling molecules. Other SPCs are known to be required during embryonic development but corresponding data regarding SPC7 have not been reported previously. Methodology/Principal Findings We demonstrated that Xenopus SPC7 (SPC7) was expressed predominantly in the developing brain and eye, throughout the neural plate initially, then more specifically in the lens and retina primordia as development progressed. Since no prior functional information has been reported for SPC7, we used gain- and loss-of-function experiments to investigate the possibility that it may also convey patterning or tissue specification information similarly to Furin, SPC4, and SPC6. Overexpression of SPC7 was without effect. In contrast, injection of SPC7 antisense morpholino oligonucleotides (MO) into a single blastomere at the 2- or 4-cell stage produced marked disruption of head structures; anophthalmia was salient. Bilateral injections suppressed head and eye formation completely. In parallel with suppression of eye and brain development by SPC7 knockdown, expression of early anterior neural markers (Sox2, Otx2, Rx2, and Pax6) and late eye-specific markers (β-Crystallin and Opsin), and of BMP target genes such as Tbx2 and Tbx3, was reduced or eliminated. Taken together, these findings suggest a critical role for SPC7–perhaps, at least in part, due to activation of one or more BMPs–in early patterning of the anterior neural plate and its derivatives. Conclusion/Significance SPC7 is required for normal development of the eye and brain, possibly through processing BMPs, though other potential substrates cannot be excluded. PMID:22761776

Biased Brownian motion as a mechanism to facilitate nanometer-scale exploration of the microtubule plus end by a kinesin-8.

PubMed

Shin, Yongdae; Du, Yaqing; Collier, Scott E; Ohi, Melanie D; Lang, Matthew J; Ohi, Ryoma

2015-07-21

Kinesin-8s are plus-end-directed motors that negatively regulate microtubule (MT) length. Well-characterized members of this subfamily (Kip3, Kif18A) exhibit two important properties: (i) They are "ultraprocessive," a feature enabled by a second MT-binding site that tethers the motors to a MT track, and (ii) they dissociate infrequently from the plus end. Together, these characteristics combined with their plus-end motility cause Kip3 and Kif18A to enrich preferentially at the plus ends of long MTs, promoting MT catastrophes or pausing. Kif18B, an understudied human kinesin-8, also limits MT growth during mitosis. In contrast to Kif18A and Kip3, localization of Kif18B to plus ends relies on binding to the plus-end tracking protein EB1, making the relationship between its potential plus-end-directed motility and plus-end accumulation unclear. Using single-molecule assays, we show that Kif18B is only modestly processive and that the motor switches frequently between directed and diffusive modes of motility. Diffusion is promoted by the tail domain, which also contains a second MT-binding site that decreases the off rate of the motor from the MT lattice. In cells, Kif18B concentrates at the extreme tip of a subset of MTs, superseding EB1. Our data demonstrate that kinesin-8 motors use diverse design principles to target MT plus ends, which likely target them to the plus ends of distinct MT subpopulations in the mitotic spindle.

Biased Brownian motion as a mechanism to facilitate nanometer-scale exploration of the microtubule plus end by a kinesin-8

PubMed Central

Shin, Yongdae; Du, Yaqing; Collier, Scott E.; Ohi, Melanie D.; Lang, Matthew J.; Ohi, Ryoma

2015-01-01

Kinesin-8s are plus-end–directed motors that negatively regulate microtubule (MT) length. Well-characterized members of this subfamily (Kip3, Kif18A) exhibit two important properties: (i) They are “ultraprocessive,” a feature enabled by a second MT-binding site that tethers the motors to a MT track, and (ii) they dissociate infrequently from the plus end. Together, these characteristics combined with their plus-end motility cause Kip3 and Kif18A to enrich preferentially at the plus ends of long MTs, promoting MT catastrophes or pausing. Kif18B, an understudied human kinesin-8, also limits MT growth during mitosis. In contrast to Kif18A and Kip3, localization of Kif18B to plus ends relies on binding to the plus-end tracking protein EB1, making the relationship between its potential plus-end–directed motility and plus-end accumulation unclear. Using single-molecule assays, we show that Kif18B is only modestly processive and that the motor switches frequently between directed and diffusive modes of motility. Diffusion is promoted by the tail domain, which also contains a second MT-binding site that decreases the off rate of the motor from the MT lattice. In cells, Kif18B concentrates at the extreme tip of a subset of MTs, superseding EB1. Our data demonstrate that kinesin-8 motors use diverse design principles to target MT plus ends, which likely target them to the plus ends of distinct MT subpopulations in the mitotic spindle. PMID:26150501

Engineered tug-of-war between kinesin and dynein controls direction of microtubule transport in vivo

PubMed Central

Rezaul, Karim; Gupta, Dipika; Semenova, Irina; Ikeda, Kazuho; Kraikivski, Pavel; Yu, Ji; Cowan, Ann; Zaliapin, Ilya; Rodionov, Vladimir

2017-01-01

Bidirectional transport of membrane organelles along microtubules (MTs) is driven by plus-end directed kinesins and minus-end directed dynein bound to the same cargo. Activities of opposing MT motors produce bidirectional movement of membrane organelles and cytoplasmic particles along MT transport tracks. Directionality of MT-based transport might be controlled by a protein complex that determines which motor type is active at any given moment of time, or determined by the outcome of a tug-of-war between MT motors dragging cargo organelles in opposite directions. However, evidence in support of each mechanisms of regulation is based mostly on the results of theoretical analyses or indirect experimental data. Here, we test whether the direction of movement of membrane organelles in vivo can be controlled by the tug-of-war between opposing MT motors alone, by attaching large number of kinesin-1 motors to organelles transported by dynein to minus-ends of MTs. We find that recruitment of kinesin significantly reduces the length and velocity of minus-end-directed dynein-dependent MT runs, leading to a reversal of the overall direction of dynein-driven organelles in vivo. Therefore in the absence of external regulators tug-of-war between opposing MT motors alone is sufficient to determine the directionality of MT transport in vivo. PMID:26843027

Transport of fungal RAB11 secretory vesicles involves myosin-5, dynein/dynactin/p25, and kinesin-1 and is independent of kinesin-3

PubMed Central

Peñalva, Miguel A.; Zhang, Jun; Xiang, Xin; Pantazopoulou, Areti

2017-01-01

Hyphal tip cells of the fungus Aspergillus nidulans are useful for studying long-range intracellular traffic. Post-Golgi secretory vesicles (SVs) containing the RAB11 orthologue RabE engage myosin-5 as well as plus end– and minus end–directed microtubule motors, providing an experimental system with which to investigate the interplay between microtubule and actin motors acting on the same cargo. By exploiting the fact that depolymerization of F-actin unleashes SVs focused at the apex by myosin-5 to microtubule-dependent motors, we establish that the minus end–directed transport of SVs requires the dynein/dynactin supercomplex. This minus end–directed transport is largely unaffected by genetic ablation of the Hook complex adapting early endosomes (EEs) to dynein but absolutely requires p25 in dynactin. Thus dynein recruitment to two different membranous cargoes, namely EEs and SVs, requires p25, highlighting the importance of the dynactin pointed-end complex to scaffold cargoes. Finally, by studying the behavior of SVs and EEs in null and rigor mutants of kinesin-3 and kinesin-1 (UncA and KinA, respectively), we demonstrate that KinA is the major kinesin mediating the anterograde transport of SVs. Therefore SVs arrive at the apex of A. nidulans by anterograde transport involving cooperation of kinesin-1 with myosin-5 and can move away from the apex powered by dynein. PMID:28209731

KCH kinesin drives nuclear transport and cytoskeletal coalescence for tip cell growth.

PubMed

Yamada, Moé; Goshima, Gohta

2018-06-07

Long-distance transport along microtubules (MTs) is critical for intracellular organisation. In animals, antagonistic motor proteins kinesin (plus end-directed) and dynein (minus end-directed) drive cargo transport. In land plants, however, the identity of motors responsible for transport is poorly understood, as genes encoding cytoplasmic dynein are absent in plant genomes. How other functions of dynein are brought about in plants also remains unknown. Here, we show that a subclass of the kinesin-14 family, KCH (kinesin with calponin homology domain)-which can also bind actin-drives MT minus end-directed nuclear transport in the moss Physcomitrella patens. When all four KCH genes were deleted, the nucleus was not maintained in the cell centre, but was translocated to the apical end of protonemal cells. In the knockout (KO) line, apical cell tip growth was also severely suppressed. KCH was localized to MTs, including at the MT focal point near the tip of protonemal cells, where MT plus ends coalesced with actin filaments. MT focus was not stably maintained in KCH KO lines, whereas actin destabilisation also disrupted the MT focus in wild-type lines despite KCH remaining on unfocused MTs. KCH had distinct functions in nuclear transport and tip growth, as a truncated KCH construct restored nuclear transport activity, but not tip growth retardation of the KO line. Thus, our study identified KCH as a long-distance retrograde transporter as well as a MT crosslinker, reminiscent of the versatile animal dynein. © 2018 American Society of Plant Biologists. All rights reserved.

Possible role of the 38 kDa protein, lacking in the gastrula-arrested Xenopus mutant, in gastrulation.

PubMed

Tanaka, Tetsuya S; Ikenishi, Kohji

2002-02-01

An acidic, 38 kDa protein that is present in Xenopus wild-type embryos has been previously shown to be lacking in gastrula-arrested mutant embryos. To gain understanding of the role of this protein, its spatio-temporal distribution and involvement in gastrulation was investigated using the monoclonal antibody (9D10) against it. The protein was prominent in the cortical cytoplasm of cells facing the outside in the animal hemisphere of embryos until the gastrula stage, and in ciliated epithelial cells of embryos at stages later than the late neurula. When the 9D10 antibody was injected into fertilized wild-type eggs, they cleaved normally, but most of them had arrested development, always at the early stage of gastrulation, as in the mutant embryos. In contrast, the majority of the control antibody-injected eggs gastrulated normally and developed further. Cytoskeletal F-actin, which was mainly observed in the area beneath the plasma membrane facing the outside of the epithelial layer of not only the dorsal involuting marginal zone but also the dorsal, vegetal cell mass of the control antibody-injected embryos at the early gastrula stage, was scarcely recognized in the corresponding area of the 9D10 antibody-injected embryos. It is likely that the paucity of the F-actin caused by the 9D10 antibody inhibition of the 38 kDa protein might lead to a failure of cell movement in gastrulation, resulting in developmental arrest.

Dynamic model of the force driving kinesin to move along microtubule-Simulation with a model system

NASA Astrophysics Data System (ADS)

Chou, Y. C.; Hsiao, Yi-Feng; To, Kiwing

2015-09-01

A dynamic model for the motility of kinesin, including stochastic-force generation and step formation is proposed. The force driving the motion of kinesin motor is generated by the impulse from the collision between the randomly moving long-chain stalk and the ratchet-shaped outer surface of microtubule. Most of the dynamical and statistical features of the motility of kinesin are reproduced in a simulation system, with (a) ratchet structures similar to the outer surface of microtubule, (b) a bead chain connected to two heads, similarly to the stalk of the real kinesin motor, and (c) the interaction between the heads of the simulated kinesin and microtubule. We also propose an experiment to discriminate between the conventional hand-over-hand model and the dynamic model.

Abelson Interactor 1 (Abi1) and Its Interaction with Wiskott-Aldrich Syndrome Protein (Wasp) Are Critical for Proper Eye Formation in Xenopus Embryos*

PubMed Central

Singh, Arvinder; Winterbottom, Emily F.; Ji, Yon Ju; Hwang, Yoo-Seok; Daar, Ira O.

2013-01-01

Abl interactor 1 (Abi1) is a scaffold protein that plays a central role in the regulation of actin cytoskeleton dynamics as a constituent of several key protein complexes, and homozygous loss of this protein leads to embryonic lethality in mice. Because this scaffold protein has been shown in cultured cells to be a critical component of pathways controlling cell migration and actin regulation at cell-cell contacts, we were interested to investigate the in vivo role of Abi1 in morphogenesis during the development of Xenopus embryos. Using morpholino-mediated translation inhibition, we demonstrate that knockdown of Abi1 in the whole embryo, or specifically in eye field progenitor cells, leads to disruption of eye morphogenesis. Moreover, signaling through the Src homology 3 domain of Abi1 is critical for proper movement of retinal progenitor cells into the eye field and their appropriate differentiation, and this process is dependent upon an interaction with the nucleation-promoting factor Wasp (Wiskott-Aldrich syndrome protein). Collectively, our data demonstrate that the Abi1 scaffold protein is an essential regulator of cell movement processes required for normal eye development in Xenopus embryos and specifically requires an Src homology 3 domain-dependent interaction with Wasp to regulate this complex morphogenetic process. PMID:23558677

Proteomic analysis of fibroblastema formation in regenerating hind limbs of Xenopus laevis froglets and comparison to axolotl

PubMed Central

2014-01-01

Background To gain insight into what differences might restrict the capacity for limb regeneration in Xenopus froglets, we used High Performance Liquid Chromatography (HPLC)/double mass spectrometry to characterize protein expression during fibroblastema formation in the amputated froglet hindlimb, and compared the results to those obtained previously for blastema formation in the axolotl limb. Results Comparison of the Xenopus fibroblastema and axolotl blastema revealed several similarities and significant differences in proteomic profiles. The most significant similarity was the strong parallel down regulation of muscle proteins and enzymes involved in carbohydrate metabolism. Regenerating Xenopus limbs differed significantly from axolotl regenerating limbs in several ways: deficiency in the inositol phosphate/diacylglycerol signaling pathway, down regulation of Wnt signaling, up regulation of extracellular matrix (ECM) proteins and proteins involved in chondrocyte differentiation, lack of expression of a key cell cycle protein, ecotropic viral integration site 5 (EVI5), that blocks mitosis in the axolotl, and the expression of several patterning proteins not seen in the axolotl that may dorsalize the fibroblastema. Conclusions We have characterized global protein expression during fibroblastema formation after amputation of the Xenopus froglet hindlimb and identified several differences that lead to signaling deficiency, failure to retard mitosis, premature chondrocyte differentiation, and failure of dorsoventral axial asymmetry. These differences point to possible interventions to improve blastema formation and pattern formation in the froglet limb. PMID:25063185

C8orf46 homolog encodes a novel protein Vexin that is required for neurogenesis in Xenopus laevis.

PubMed

Moore, Kathryn B; Logan, Mary A; Aldiri, Issam; Roberts, Jacqueline M; Steele, Michael; Vetter, Monica L

2018-05-01

Neural basic helix-loop helix (bHLH) transcription factors promote progenitor cell differentiation by activation of downstream target genes that coordinate neuronal differentiation. Here we characterize a neural bHLH target gene in Xenopus laevis, vexin (vxn; previously sbt1), that is homologous to human c8orf46 and is conserved across vertebrate species. C8orf46 has been implicated in cancer progression, but its function is unknown. Vxn is transiently expressed in differentiating progenitors in the developing central nervous system (CNS), and is required for neurogenesis in the neural plate and retina. Its function is conserved, since overexpression of either Xenopus or mouse vxn expands primary neurogenesis and promotes early retinal cell differentiation in cooperation with neural bHLH factors. Vxn protein is localized to the cell membrane and the nucleus, but functions in the nucleus to promote neural differentiation. Vxn inhibits cell proliferation, and works with the cyclin-dependent kinase inhibitor p27Xic1 (cdkn1b) to enhance neurogenesis and increase levels of the proneural protein Neurog2. We propose that vxn provides a key link between neural bHLH activity and execution of the neurogenic program. Copyright © 2018 Elsevier Inc. All rights reserved.

The Xenopus Tgfbi is required for embryogenesis through regulation of canonical Wnt signalling.

PubMed

Wang, Feng; Hu, Wanzhou; Xian, Jian; Ohnuma, Shin-ichi; Brenton, James D

2013-07-01

Tgfbi, a fasciclin family extracellular matrix protein, has various roles in human diseases from corneal dystrophies to cancer. However, the molecular mechanisms that underlie its functions are poorly understood. Here, we studied the role of Tgfbi during Xenopus embryogenesis. During gastrulation and immediately after, Xtgfbi is expressed at developmentally important signaling centers including the dorsal marginal zone, notochord and floorplate. Xtgfbi knockdown by anti-sense morpholinos causes defective organizer induction, patterning and differentiation of muscle, neuron and neural crests, similar to suppression of canonical Wnt signaling. In Xenopus embryos and animal caps as well as DLD-1 cells, we show that Tgfbi is strongly required for the full activation of the canonical Wnt pathway by promoting phosphorylation of GSK3β and consequently enhancing the stabilization and nuclear localization of β-catenin. Further analysis shows that Tgfbi is likely to promote GSK3β phosphorylation through integrin-linked kinase. Copyright © 2013 Elsevier Inc. All rights reserved.

Class I HDACs control a JIP1-dependent pathway for kinesin-microtubule binding in cardiomyocytes

PubMed Central

Blakeslee, Weston W.; Lin, Ying-Hsi; Stratton, Matthew S.; Tatman, Philip D.; Hu, Tianjing; Ferguson, Bradley S.; McKinsey, Timothy A.

2018-01-01

Class I histone deacetylase (HDAC) inhibitors block hypertrophy and fibrosis of the heart by suppressing pathological signaling and gene expression programs in cardiac myocytes and fibroblasts. The impact of HDAC inhibition in unstressed cardiac cells remains poorly understood. Here, we demonstrate that treatment of cultured cardiomyocytes with small molecule HDAC inhibitors leads to dramatic induction of c-Jun amino-terminal kinase (JNK)-interacting protein-1 (JIP1) mRNA and protein expression. In contrast to prior findings, elevated levels of endogenous JIP1 in cardiomyocytes failed to significantly alter JNK signaling or cardiomyocyte hypertrophy. Instead, HDAC inhibitor-mediated induction of JIP1 was required to stimulate expression of the kinesin heavy chain family member, KIF5A. We provide evidence for an HDAC-dependent regulatory circuit that promotes formation of JIP1:KIF5A:microtubule complexes that regulate intracellular transport of cargo such as autophagosomes. These findings define a novel role for class I HDACs in the control of the JIP1/kinesin axis in cardiomyocytes, and suggest that HDAC inhibitors could be used to alter microtubule transport in the heart. PMID:28886967

Kinesin-5-independent mitotic spindle assembly requires the antiparallel microtubule crosslinker Ase1 in fission yeast

PubMed Central

Rincon, Sergio A.; Lamson, Adam; Blackwell, Robert; Syrovatkina, Viktoriya; Fraisier, Vincent; Paoletti, Anne; Betterton, Meredith D.; Tran, Phong T.

2017-01-01

Bipolar spindle assembly requires a balance of forces where kinesin-5 produces outward pushing forces to antagonize the inward pulling forces from kinesin-14 or dynein. Accordingly, Kinesin-5 inactivation results in force imbalance leading to monopolar spindle and chromosome segregation failure. In fission yeast, force balance is restored when both kinesin-5 Cut7 and kinesin-14 Pkl1 are deleted, restoring spindle bipolarity. Here we show that the cut7Δpkl1Δ spindle is fully competent for chromosome segregation independently of motor activity, except for kinesin-6 Klp9, which is required for anaphase spindle elongation. We demonstrate that cut7Δpkl1Δ spindle bipolarity requires the microtubule antiparallel bundler PRC1/Ase1 to recruit CLASP/Cls1 to stabilize microtubules. Brownian dynamics-kinetic Monte Carlo simulations show that Ase1 and Cls1 activity are sufficient for initial bipolar spindle formation. We conclude that pushing forces generated by microtubule polymerization are sufficient to promote spindle pole separation and the assembly of bipolar spindle in the absence of molecular motors. PMID:28513584

Kinesin Motor Enzymology: Chemistry, Structure, and Physics of Nanoscale Molecular Machines.

PubMed

Cochran, J C

2015-09-01

Molecular motors are enzymes that convert chemical potential energy into controlled kinetic energy for mechanical work inside cells. Understanding the biophysics of these motors is essential for appreciating life as well as apprehending diseases that arise from motor malfunction. This review focuses on kinesin motor enzymology with special emphasis on the literature that reports the chemistry, structure and physics of several different kinesin superfamily members.

Musashi and Plasticity of Xenopus and Axolotl Spinal Cord Ependymal Cells

PubMed Central

Chernoff, Ellen A. G.; Sato, Kazuna; Salfity, Hai V. N.; Sarria, Deborah A.; Belecky-Adams, Teri

2018-01-01

The differentiated state of spinal cord ependymal cells in regeneration-competent amphibians varies between a constitutively active state in what is essentially a developing organism, the tadpole of the frog Xenopus laevis, and a quiescent, activatable state in a slowly growing adult salamander Ambystoma mexicanum, the Axolotl. Ependymal cells are epithelial in intact spinal cord of all vertebrates. After transection, body region ependymal epithelium in both Xenopus and the Axolotl disorganizes for regenerative outgrowth (gap replacement). Injury-reactive ependymal cells serve as a stem/progenitor cell population in regeneration and reconstruct the central canal. Expression patterns of mRNA and protein for the stem/progenitor cell-maintenance Notch signaling pathway mRNA-binding protein Musashi (msi) change with life stage and regeneration competence. Msi-1 is missing (immunohistochemistry), or at very low levels (polymerase chain reaction, PCR), in both intact regeneration-competent adult Axolotl cord and intact non-regeneration-competent Xenopus tadpole (Nieuwkoop and Faber stage 62+, NF 62+). The critical correlation for successful regeneration is msi-1 expression/upregulation after injury in the ependymal outgrowth and stump-region ependymal cells. msi-1 and msi-2 isoforms were cloned for the Axolotl as well as previously unknown isoforms of Xenopus msi-2. Intact Xenopus spinal cord ependymal cells show a loss of msi-1 expression between regeneration-competent (NF 50–53) and non-regenerating stages (NF 62+) and in post-metamorphosis froglets, while msi-2 displays a lower molecular weight isoform in non-regenerating cord. In the Axolotl, embryos and juveniles maintain Msi-1 expression in the intact cord. In the adult Axolotl, Msi-1 is absent, but upregulates after injury. Msi-2 levels are more variable among Axolotl life stages: rising between late tailbud embryos and juveniles and decreasing in adult cord. Cultures of regeneration-competent Xenopus tadpole

G protein-coupled receptors Flop1 and Flop2 inhibit Wnt/β-catenin signaling and are essential for head formation in Xenopus.

PubMed

Miyagi, Asuka; Negishi, Takefumi; Yamamoto, Takamasa S; Ueno, Naoto

2015-11-01

Patterning of the vertebrate anterior-posterior axis is regulated by the coordinated action of growth factors whose effects can be further modulated by upstream and downstream mediators and the cross-talk of different intracellular pathways. In particular, the inhibition of the Wnt/β-catenin signaling pathway by various factors is critically required for anterior specification. Here, we report that Flop1 and Flop2 (Flop1/2), G protein-coupled receptors related to Gpr4, contribute to the regulation of head formation by inhibiting Wnt/β-catenin signaling in Xenopus embryos. Using whole-mount in situ hybridization, we showed that flop1 and flop2 mRNAs were expressed in the neural ectoderm during early gastrulation. Both the overexpression and knockdown of Flop1/2 resulted in altered embryonic head phenotypes, while the overexpression of either Flop1/2 or the small GTPase RhoA in the absence of bone morphogenetic protein (BMP) signaling resulted in ectopic head induction. Examination of the Flops' function in Xenopus embryo animal cap cells showed that they inhibited Wnt/β-catenin signaling by promoting β-catenin degradation through both RhoA-dependent and -independent pathways in a cell-autonomous manner. These results suggest that Flop1 and Flop2 are essential regulators of Xenopus head formation that act as novel inhibitory components of the Wnt/β-catenin signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

Effective killing of the human pathogen Candida albicans by a specific inhibitor of non-essential mitotic kinesin Kip1p

PubMed Central

Chua, Penelope R; Roof, David M; Lee, Yan; Sakowicz, Roman; Clarke, David; Pierce, Dan; Stephens, Thoryn; Hamilton, Matthew; Morgan, Brad; Morgans, David; Nakai, Takashi; Tomasi, Adam; Maxon, Mary E

2007-01-01

Kinesins from the bipolar (Kinesin-5) family are conserved in eukaryotic organisms and play critical roles during the earliest stages of mitosis to mediate spindle pole body separation and formation of a bipolar mitotic spindle. To date, genes encoding bipolar kinesins have been reported to be essential in all organisms studied. We report the characterization of CaKip1p, the sole member of this family in the human pathogenic yeast Candida albicans. C. albicans Kip1p appears to localize to the mitotic spindle and loss of CaKip1p function interferes with normal progression through mitosis. Inducible excision of CaKIP1 revealed phenotypes unique to C. albicans, including viable homozygous Cakip1 mutants and an aberrant spindle morphology in which multiple spindle poles accumulate in close proximity to each other. Expression of the C. albicans Kip1 motor domain in Escherichia coli produced a protein with microtubule-stimulated ATPase activity that was inhibited by an aminobenzothiazole (ABT) compound in an ATP-competitive fashion. This inhibition results in ‘rigor-like’, tight association with microtubules in vitro. Upon treatment of C. albicans cells with the ABT compound, cells were killed, and terminal phenotype analysis revealed an aberrant spindle morphology similar to that induced by loss of the CaKIP1 gene. The ABT compound discovered is the first example of a fungal spindle inhibitor targeted to a mitotic kinesin. Our results also show that the non-essential nature and implementation of the bipolar motor in C. albicans differs from that seen in other organisms, and suggest that inhibitors of a non-essential mitotic kinesin may offer promise as cidal agents for antifungal drug discovery. PMID:17573815

[Crystallography of ATP hydrolysis mechanism in rat brain kinesin].

PubMed

Wan, Qun; Zhu, Pingting; Lü, Houning; Chen, Xinhong

2014-04-01

Rat brain kinesin is a conventional kinesin that uses the energy from ATP hydrolysis to walk along the microtubule progressively. Studying how the chemical energy in ATP is utilized for mechanical movement is important to understand this moving function. The monomeric motor domain, rK354, was crystallized. An ATP analog, AMPPNP, was soaked in the active site. Comparing the complex structure of rK354 x AMPPNP and that of rK354ADP, a hypothesis is proposed that Glu237 in the Switch II region sensors the presence of gamma-phosphate and transfers the signal to the microtubule binding region.

Depletion force induced collective motion of microtubules driven by kinesin

NASA Astrophysics Data System (ADS)

Inoue, Daisuke; Mahmot, Bulbul; Kabir, Arif Md. Rashedul; Farhana, Tamanna Ishrat; Tokuraku, Kiyotaka; Sada, Kazuki; Konagaya, Akihiko; Kakugo, Akira

2015-10-01

Collective motion is a fascinating example of coordinated behavior of self-propelled objects, which is often associated with the formation of large scale patterns. Nowadays, the in vitro gliding assay is being considered a model system to experimentally investigate various aspects of group behavior and pattern formation by self-propelled objects. In the in vitro gliding assay, cytoskeletal filaments F-actin or microtubules are driven by the surface immobilized associated biomolecular motors myosin or dynein respectively. Although the F-actin/myosin or microtubule/dynein system was found to be promising in understanding the collective motion and pattern formation by self-propelled objects, the most widely used biomolecular motor system microtubule/kinesin could not be successfully employed so far in this regard. Failure in exhibiting collective motion by kinesin driven microtubules is attributed to the intrinsic properties of kinesin, which was speculated to affect the behavior of individual gliding microtubules and mutual interactions among them. In this work, for the first time, we have demonstrated the collective motion of kinesin driven microtubules by regulating the mutual interaction among the gliding microtubules, by employing a depletion force among them. Proper regulation of the mutual interaction among the gliding microtubules through the employment of the depletion force was found to allow the exhibition of collective motion and stream pattern formation by the microtubules. This work offers a universal means for demonstrating the collective motion using the in vitro gliding assay of biomolecular motor systems and will help obtain a meticulous understanding of the fascinating coordinated behavior and pattern formation by self-propelled objects.Collective motion is a fascinating example of coordinated behavior of self-propelled objects, which is often associated with the formation of large scale patterns. Nowadays, the in vitro gliding assay is being

Decrypting the structural, dynamic, and energetic basis of a monomeric kinesin interacting with a tubulin dimer in three ATPase states by all-atom molecular dynamics simulation.

PubMed

Chakraborty, Srirupa; Zheng, Wenjun

2015-01-27

We have employed molecular dynamics (MD) simulation to investigate, with atomic details, the structural dynamics and energetics of three major ATPase states (ADP, APO, and ATP state) of a human kinesin-1 monomer in complex with a tubulin dimer. Starting from a recently solved crystal structure of ATP-like kinesin-tubulin complex by the Knossow lab, we have used flexible fitting of cryo-electron-microscopy maps to construct new structural models of the kinesin-tubulin complex in APO and ATP state, and then conducted extensive MD simulations (total 400 ns for each state), followed by flexibility analysis, principal component analysis, hydrogen bond analysis, and binding free energy analysis. Our modeling and simulation have revealed key nucleotide-dependent changes in the structure and flexibility of the nucleotide-binding pocket (featuring a highly flexible and open switch I in APO state) and the tubulin-binding site, and allosterically coupled motions driving the APO to ATP transition. In addition, our binding free energy analysis has identified a set of key residues involved in kinesin-tubulin binding. On the basis of our simulation, we have attempted to address several outstanding issues in kinesin study, including the possible roles of β-sheet twist and neck linker docking in regulating nucleotide release and binding, the structural mechanism of ADP release, and possible extension and shortening of α4 helix during the ATPase cycle. This study has provided a comprehensive structural and dynamic picture of kinesin's major ATPase states, and offered promising targets for future mutational and functional studies to investigate the molecular mechanism of kinesin motors.

Nucleotide sequence of the L1 ribosomal protein gene of Xenopus laevis: remarkable sequence homology among introns.

PubMed Central

Loreni, F; Ruberti, I; Bozzoni, I; Pierandrei-Amaldi, P; Amaldi, F

1985-01-01

Ribosomal protein L1 is encoded by two genes in Xenopus laevis. The comparison of two cDNA sequences shows that the two L1 gene copies (L1a and L1b) have diverged in many silent sites and very few substitution sites; moreover a small duplication occurred at the very end of the coding region of the L1b gene which thus codes for a product five amino acids longer than that coded by L1a. Quantitatively the divergence between the two L1 genes confirms that a whole genome duplication took place in Xenopus laevis approximately 30 million years ago. A genomic fragment containing one of the two L1 gene copies (L1a), with its nine introns and flanking regions, has been completely sequenced. The 5' end of this gene has been mapped within a 20-pyridimine stretch as already found for other vertebrate ribosomal protein genes. Four of the nine introns have a 60-nucleotide sequence with 80% homology; within this region some boxes, one of which is 16 nucleotides long, are 100% homologous among the four introns. This feature of L1a gene introns is interesting since we have previously shown that the activity of this gene is regulated at a post-transcriptional level and it involves the block of the normal splicing of some intron sequences. Images Fig. 3. Fig. 5. PMID:3841512

Binding to membrane proteins within the endoplasmic reticulum cannot explain the retention of the glucose-regulated protein GRP78 in Xenopus oocytes.

PubMed

Ceriotti, A; Colman, A

1988-03-01

We have studied the compartmentation and movement of the rat 78-kd glucose-regulated protein (GRP78) and other secretory and membrane proteins in Xenopus oocytes. Full length GRP78, normally found in the lumen of rat endoplasmic reticulum (ER), is localized to a membraneous compartment in oocytes and is not secreted. A truncated GRP78 lacking the C-terminal (KDEL) ER retention signal is secreted, although at a slow rate. When the synthesis of radioactive GRP78 is confined to a polar (animal or vegetal) region of the oocyte and the subsequent movement across the oocyte monitored, we find that both full-length and truncated GRP78 move at similar rates and only slightly slower than a secretory protein, chick ovalbumin. In contrast, a plasma membrane protein (influenza haemagglutinin) and two ER membrane proteins (rotavirus VP10 and a mutant haemagglutinin) remained confined to their site of synthesis. We conclude that the retention of GRP78 in the ER is not due to its tight binding to a membrane-bound receptor.

Sequencing and analysis of 10967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morin, R D; Chang, E; Petrescu, A

2005-10-31

Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection initiative. Here we present an analysis of 10967 clones (8049 from X. laevis and 2918 from X. tropicalis). The clone set contains 2013 orthologs between X. laevis and X. tropicalis as well as 1795 paralog pairs within X. laevis. 1199 are in-paralogs, believed to have resulted from an allotetraploidization event approximately 30 million years ago, and the remaining 546 are likely out-paralogs that have resulted from more ancient gene duplications, prior to the divergence betweenmore » the two species. We do not detect any evidence for positive selection by the Yang and Nielsen maximum likelihood method of approximating d{sub N}/d{sub S}. However, d{sub N}/d{sub S} for X. laevis in-paralogs is elevated relative to X. tropicalis orthologs. This difference is highly significant, and indicates an overall relaxation of selective pressures on duplicated gene pairs. Within both groups of paralogs, we found evidence of subfunctionalization, manifested as differential expression of paralogous genes among tissues, as measured by EST information from public resources. We have observed, as expected, a higher instance of subfunctionalization in out-paralogs relative to in-paralogs.« less

Ski represses BMP signaling in Xenopus and mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

kluo@lbl.gov

2001-05-16

The bone morphogenic proteins (BMPs) play important roles in vertebrate development. In Xenopus, BMPs act as epidermal inducers and also as negative regulators of neurogenesis. Antagonism of BMP signaling results in neuralization. BMPs signal through the cell-surface receptors and downstream Smad molecules. Upon stimulation with BMP, Smad1, Smad5, and Smad8 are phosphorylated by the activated BMP receptors, form a complex with Smad4, and translocate into the nucleus, where they regulate the expression of BMP target genes. Here, we show that the Ski oncoprotein can block BMP signaling and the expression of BMP-responsive genes in both Xenopus and mammalian cells bymore » directly interacting with and repressing the activity of BMP-specific Smad complexes. This ability to antagonize BMP signaling results in neuralization by Ski in the Xenopus embryo and blocking of osteoblast differentiation of murine W-20-17 cells. Thus, Ski is able to repress the activity of all receptor-associated Smads and may regulate vertebrate development by modulating the signaling activity of transforming growth factor-{beta} family members.« less

Triphenylbutanamines: Kinesin Spindle Protein Inhibitors with in Vivo Antitumor Activity†

PubMed Central

2012-01-01

The human mitotic kinesin Eg5 represents a novel mitotic spindle target for cancer chemotherapy. We previously identified S-trityl-l-cysteine (STLC) and related analogues as selective potent inhibitors of Eg5. We herein report on the development of a series of 4,4,4-triphenylbutan-1-amine inhibitors derived from the STLC scaffold. This new generation systematically improves on potency: the most potent C-trityl analogues exhibit Kiapp ≤ 10 nM and GI50 ≈ 50 nM, comparable to results from the phase II clinical benchmark ispinesib. Crystallographic studies reveal that they adopt the same overall binding configuration as S-trityl analogues at an allosteric site formed by loop L5 of Eg5. Evaluation of their druglike properties reveals favorable profiles for future development and, in the clinical candidate ispinesib, moderate hERG and CYP inhibition. One triphenylbutanamine analogue and ispinesib possess very good bioavailability (51% and 45%, respectively), with the former showing in vivo antitumor growth activity in nude mice xenograft studies. PMID:22248262

Antisense RNA to the first N-glycosylation gene, ALG7, inhibits protein N-glycosylation and secretion by Xenopus oocytes.

PubMed

Kukuruzinska, M A; Apekin, V; Lamkin, M S; Hiltz, A; Rodriguez, A; Lin, C C; Paz, M A; Oppenheim, F G

1994-02-15

N-Glycosylation has been shown to affect the rate of glycoprotein transport through the secretory pathway. In order to identify the critical components in the N-glycosylation pathway that directly influence protein secretion, we have studied the effects of downregulation of the first gene in the dolichol pathway, ALG7, on the synthesis, glycosylation and secretion of native and heterologous proteins by Xenopus laevis oocytes. Our strategy involved the use of ALG7 antisense RNA (asRNA) to lower the effective abundance of the ALG7 protein in oocytes. The results showed that there was an inverse dose-response relationship between ALG7 asRNA and the amount of glycosylated and secreted proteins. These effects were also observed for heterologously expressed rat parotid amylase. Since ALG7 asRNA did not inhibit overall protein synthesis, we conclude that downregulation of ALG7 expression directly lowered protein export.

BORC Functions Upstream of Kinesins 1 and 3 to Coordinate Regional Movement of Lysosomes along Different Microtubule Tracks.

PubMed

Guardia, Carlos M; Farías, Ginny G; Jia, Rui; Pu, Jing; Bonifacino, Juan S

2016-11-15

The multiple functions of lysosomes are critically dependent on their ability to undergo bidirectional movement along microtubules between the center and the periphery of the cell. Centrifugal and centripetal movement of lysosomes is mediated by kinesin and dynein motors, respectively. We recently described a multi-subunit complex named BORC that recruits the small GTPase Arl8 to lysosomes to promote their kinesin-dependent movement toward the cell periphery. Here, we show that BORC and Arl8 function upstream of two structurally distinct kinesin types: kinesin-1 (KIF5B) and kinesin-3 (KIF1Bβ and KIF1A). Remarkably, KIF5B preferentially moves lysosomes on perinuclear tracks enriched in acetylated α-tubulin, whereas KIF1Bβ and KIF1A drive lysosome movement on more rectilinear, peripheral tracks enriched in tyrosinated α-tubulin. These findings establish BORC as a master regulator of lysosome positioning through coupling to different kinesins and microtubule tracks. Common regulation by BORC enables coordinate control of lysosome movement in different regions of the cell. Published by Elsevier Inc.

BORC Functions Upstream of Kinesins 1 and 3 to Coordinate Regional Movement of Lysosomes Along Different Microtubule Tracks

PubMed Central

Guardia, Carlos M.; Farías, Ginny G.; Jia, Rui; Pu, Jing; Bonifacino, Juan S.

2016-01-01

Summary The multiple functions of lysosomes are critically dependent on their ability to undergo bidirectional movement along microtubules between the center and the periphery of the cell. Centrifugal and centripetal movement of lysosomes is mediated by kinesin and dynein motors, respectively. We recently described a multisubunit complex named BORC that recruits the small GTPase Arl8 to lysosomes to promote their kinesin-dependent movement toward the cell periphery. Here we show that BORC and Arl8 function upstream of two structurally distinct kinesin types: kinesin-1 (KIF5B) and kinesin-3 (KIF1Bβ and KIF1A). Remarkably, KIF5B preferentially moves lysosomes on perinuclear tracks enriched in acetylated α-tubulin, whereas KIF1Bβ and KIF1A drive lysosome movement on more rectilinear, peripheral tracks enriched in tyrosinated α-tubulin. These findings establish BORC as a master regulator of lysosome positioning through coupling to different kinesins and microtubule tracks. Common regulation by BORC enables coordinate control of lysosome movement in different regions of the cell. PMID:27851960

The Phragmoplast-Orienting Kinesin-12 Class Proteins Translate the Positional Information of the Preprophase Band to Establish the Cortical Division Zone in Arabidopsis thaliana[C][W

PubMed Central

Lipka, Elisabeth; Gadeyne, Astrid; Stöckle, Dorothee; Zimmermann, Steffi; De Jaeger, Geert; Ehrhardt, David W.; Kirik, Viktor; Van Damme, Daniel; Müller, Sabine

2014-01-01

The preprophase band (PPB) is a faithful but transient predictor of the division plane in somatic cell divisions. Throughout mitosis the PPBs positional information is preserved by factors that continuously mark the division plane at the cell cortex, the cortical division zone, by their distinct spatio-temporal localization patterns. However, the mechanism maintaining these identity factors at the plasma membrane after PPB disassembly remains obscure. The pair of kinesin-12 class proteins PHRAGMOPLAST ORIENTING KINESIN1 (POK1) and POK2 are key players in division plane maintenance. Here, we show that POK1 is continuously present at the cell cortex, providing a spatial reference for the site formerly occupied by the PPB. Fluorescence recovery after photobleaching analysis combined with microtubule destabilization revealed dynamic microtubule-dependent recruitment of POK1 to the PPB during prophase, while POK1 retention at the cortical division zone in the absence of cortical microtubules appeared static. POK function is strictly required to maintain the division plane identity factor TANGLED (TAN) after PPB disassembly, although POK1 and TAN recruitment to the PPB occur independently during prophase. Together, our data suggest that POKs represent fundamental early anchoring components of the cortical division zone, translating and preserving the positional information of the PPB by maintaining downstream identity markers. PMID:24972597

Kinesin-8 Motors Improve Nuclear Centering by Promoting Microtubule Catastrophe

NASA Astrophysics Data System (ADS)

Glunčić, Matko; Maghelli, Nicola; Krull, Alexander; Krstić, Vladimir; Ramunno-Johnson, Damien; Pavin, Nenad; Tolić, Iva M.

2015-02-01

In fission yeast, microtubules push against the cell edge, thereby positioning the nucleus in the cell center. Kinesin-8 motors regulate microtubule catastrophe; however, their role in nuclear positioning is not known. Here we develop a physical model that describes how kinesin-8 motors affect nuclear centering by promoting a microtubule catastrophe. Our model predicts the improved centering of the nucleus in the presence of motors, which we confirmed experimentally in living cells. The model also predicts a characteristic time for the recentering of a displaced nucleus, which is supported by our experiments where we displaced the nucleus using optical tweezers.

p120 catenin associates with kinesin and facilitates the transport of cadherin–catenin complexes to intercellular junctions

PubMed Central

Chen, Xinyu; Kojima, Shin-ichiro; Borisy, Gary G.; Green, Kathleen J.

2003-01-01

p120 catenin (p120) is a component of adherens junctions and has been implicated in regulating cadherin-based cell adhesion as well as the activity of Rho small GTPases, but its exact roles in cell–cell adhesion are unclear. Using time-lapse imaging, we show that p120-GFP associates with vesicles and exhibits unidirectional movements along microtubules. Furthermore, p120 forms a complex with kinesin heavy chain through the p120 NH2-terminal head domain. Overexpression of p120, but not an NH2-terminal deletion mutant deficient in kinesin binding, recruits endogenous kinesin to N-cadherin. Disruption of the interaction between N-cadherin and p120, or the interaction between p120 and kinesin, leads to a delayed accumulation of N-cadherin at cell–cell contacts during calcium-initiated junction reassembly. Our analyses identify a novel role of p120 in promoting cell surface trafficking of cadherins via association and recruitment of kinesin. PMID:14610057

Clustered Xenopus keratin genes: A genomic, transcriptomic, and proteomic analysis.

PubMed

Suzuki, Ken-Ichi T; Suzuki, Miyuki; Shigeta, Mitsuki; Fortriede, Joshua D; Takahashi, Shuji; Mawaribuchi, Shuuji; Yamamoto, Takashi; Taira, Masanori; Fukui, Akimasa

2017-06-15

Keratin genes belong to the intermediate filament superfamily and their expression is altered following morphological and physiological changes in vertebrate epithelial cells. Keratin genes are divided into two groups, type I and II, and are clustered on vertebrate genomes, including those of Xenopus species. Various keratin genes have been identified and characterized by their unique expression patterns throughout ontogeny in Xenopus laevis; however, compilation of previously reported and newly identified keratin genes in two Xenopus species is required for our further understanding of keratin gene evolution, not only in amphibians but also in all terrestrial vertebrates. In this study, 120 putative type I and II keratin genes in total were identified based on the genome data from two Xenopus species. We revealed that most of these genes are highly clustered on two homeologous chromosomes, XLA9_10 and XLA2 in X. laevis, and XTR10 and XTR2 in X. tropicalis, which are orthologous to those of human, showing conserved synteny among tetrapods. RNA-Seq data from various embryonic stages and adult tissues highlighted the unique expression profiles of orthologous and homeologous keratin genes in developmental stage- and tissue-specific manners. Moreover, we identified dozens of epidermal keratin proteins from the whole embryo, larval skin, tail, and adult skin using shotgun proteomics. In light of our results, we discuss the radiation, diversification, and unique expression of the clustered keratin genes, which are closely related to epidermal development and terrestrial adaptation during amphibian evolution, including Xenopus speciation. Copyright © 2016 Elsevier Inc. All rights reserved.

Cell lineage tracing during Xenopus tail regeneration.

PubMed

Gargioli, Cesare; Slack, Jonathan M W

2004-06-01

The tail of the Xenopus tadpole will regenerate following amputation, and all three of the main axial structures - the spinal cord, the notochord and the segmented myotomes - are found in the regenerated tail. We have investigated the cellular origin of each of these three tissue types during regeneration. We produced Xenopus laevis embryos transgenic for the CMV (Simian Cytomegalovirus) promoter driving GFP (Green Fluorescent Protein) ubiquitously throughout the embryo. Single tissues were then specifically labelled by making grafts at the neurula stage from transgenic donors to unlabelled hosts. When the hosts have developed to tadpoles, they carry a region of the appropriate tissue labelled with GFP. These tails were amputated through the labelled region and the distribution of labelled cells in the regenerate was followed. We also labelled myofibres using the Cre-lox method. The results show that the spinal cord and the notochord regenerate from the same tissue type in the stump, with no labelling of other tissues. In the case of the muscle, we show that the myofibres of the regenerate arise from satellite cells and not from the pre-existing myofibres. This shows that metaplasia between differentiated cell types does not occur, and that the process of Xenopus tail regeneration is more akin to tissue renewal in mammals than to urodele tail regeneration.

Controlling self-assembly of microtubule spools via kinesin motor density

PubMed Central

Lam, A.T.; Curschellas, C.; Krovvidi, D.; Hess, H.

2014-01-01

Active self-assembly, in which non-thermal energy is consumed by the system to put together building blocks, allows the creation of non-equilibrium structures and active materials. Microtubule spools assembled in gliding assays are one example of such non-equilibrium structures, capable of storing bending energies on the order of 105 kT. Although these structures arise spontaneously in experiments, the origin of microtubule spooling has long been debated. Here, using a stepwise kinesin gradient, we demonstrate that spool assembly can be controlled by the surface density of kinesin motors, showing that pinning of microtubules due to dead motors plays a dominant role in spool initiation. PMID:25269076

Controlling self-assembly of microtubule spools via kinesin motor density.

PubMed

Lam, A T; Curschellas, C; Krovvidi, D; Hess, H

2014-11-21

Active self-assembly, in which non-thermal energy is consumed by the system to put together building blocks, allows the creation of non-equilibrium structures and active materials. Microtubule spools assembled in gliding assays are one example of such non-equilibrium structures, capable of storing bending energies on the order of 10(5) kT. Although these structures arise spontaneously in experiments, the origin of microtubule spooling has long been debated. Here, using a stepwise kinesin gradient, we demonstrate that spool assembly can be controlled by the surface density of kinesin motors, showing that pinning of microtubules due to dead motors plays a dominant role in spool initiation.

Effect of hemin, baicalein and heme oxygenase-1 (HO-1) enzyme activity inhibitors on Cd-induced accumulation of HO-1, HSPs and aggresome-like structures in Xenopus kidney epithelial cells.

PubMed

Campbell, James H; Heikkila, John J

2018-04-23

Cadmium is a highly toxic environmental pollutant that can cause many adverse effects including cancer, neurological disease and kidney damage. Aquatic amphibians are particularly susceptible to this toxicant as it was shown to cause developmental abnormalities and genotoxic effects. In mammalian cells, the accumulation of heme oxygenase-1 (HO-1), which catalyzes the breakdown of heme into CO, free iron and biliverdin, was reported to protect cells against potentially lethal concentrations of CdCl 2 . In the present study, CdCl 2 treatment of A6 kidney epithelial cells, derived from the frog, Xenopus laevis, induced the accumulation of HO-1, heat shock protein 70 (HSP70) and HSP30 as well as an increase in the production of aggregated protein and aggresome-like structures. Treatment of cells with inhibitors of HO-1 enzyme activity, tin protoporphyrin (SnPP) and zinc protoporphyrin (ZnPP), enhanced CdCl 2 -induced actin cytoskeletal disorganization and the accumulation of HO-1, HSP70, aggregated protein and aggresome-like structures. Treatment of cells with hemin and baicalein, which were previously shown to provide cytoprotection against various stresses, induced HO-1 accumulation in a concentration-dependent manner. Also, treatment of cells with hemin and baicalein suppressed CdCl 2 -induced actin dysregulation and the accumulation of aggregated protein and aggresome-like structures. This cytoprotective effect was inhibited by SnPP. These results suggest that HO-1-mediated protection against CdCl 2 toxicity includes the maintenance of actin cytoskeletal and microtubular structure and the suppression of aggregated protein and aggresome-like structures. Copyright © 2018 Elsevier Inc. All rights reserved.

Membrane transport of WAVE2 and lamellipodia formation require Pak1 that mediates phosphorylation and recruitment of stathmin/Op18 to Pak1-WAVE2-kinesin complex.

PubMed

Takahashi, Kazuhide; Suzuki, Katsuo

2009-05-01

Membrane transport of WAVE2 that leads to lamellipodia formation requires a small GTPase Rac1, the motor protein kinesin, and microtubules. Here we explore the possibility of whether the Rac1-dependent and kinesin-mediated WAVE2 transport along microtubules is regulated by a p21-activated kinase Pak as a downstream effector of Rac1. We find that Pak1 constitutively binds to WAVE2 and is transported with WAVE2 to the leading edge by stimulation with hepatocyte growth factor (HGF). Concomitantly, phosphorylation of tubulin-bound stathmin/Op18 at serine 25 (Ser25) and Ser38, microtubule growth, and stathmin/Op18 binding to kinesin-WAVE2 complex were induced. The HGF-induced WAVE2 transport, lamellipodia formation, stathmin/Op18 phosphorylation at Ser38 and binding to kinesin-WAVE2 complex, but not stathmin/Op18 phosphorylation at Ser25 and microtubule growth, were abrogated by Pak1 inhibitor IPA-3 and Pak1 depletion with small interfering RNA (siRNA). Moreover, stathmin/Op18 depletion with siRNA caused significant inhibition of HGF-induced WAVE2 transport and lamellipodia formation, with HGF-independent promotion of microtubule growth. Collectively, it is suggested that Pak1 plays a critical role in HGF-induced WAVE2 transport and lamellipodia formation by directing Pak1-WAVE2-kinesin complex toward the ends of growing microtubules through phosphorylation and recruitment of tubulin-bound stathmin/Op18 to the complex.

KIFC1-Like Motor Protein Associates with the Cephalopod Manchette and Participates in Sperm Nuclear Morphogenesis in Octopus tankahkeei

PubMed Central

Tan, Fu-Qing; Yang, Wan-Xi

2010-01-01

Background Nuclear morphogenesis is one of the most fundamental cellular transformations taking place during spermatogenesis. In rodents, a microtubule-based perinuclear structure, the manchette, and a C-terminal kinesin motor KIFC1 are believed to play crucial roles in this process. Spermatogenesis in Octopus tankahkeei is a good model system to explore whether evolution has created a cephalopod prototype of mammalian manchette-based and KIFC1-dependent sperm nuclear shaping machinery. Methodology/Principal Findings We detected the presence of a KIFC1-like protein in the testis, muscle, and liver of O. tankahkeei by Western Blot. Then we tracked its dynamic localization in spermatic cells at various stages using Immunofluorescence and Immunogold Electron Microscopy. The KIFC1-like protein was not expressed at early stages of spermatogenesis when no significant morphological changes occur, began to be present in early spermatid, localized around and in the nucleus of intermediate and late spermatids where the nucleus was dramatically elongated and compressed, and concentrated at one end of final spermatid. Furthermore, distribution of the motor protein during nuclear elongation and condensation overlapped with that of the cephalopod counterpart of manchette at a significant level. Conclusions/Significance The results support the assumption that the protein is actively involved in sperm nuclear morphogenesis in O. tankahkeei possibly through bridging the manchette-like perinuclear microtubules to the nucleus and assisting in the nucleocytoplasmic trafficking of specific cargoes. This study represents the first description of the role of a motor protein in sperm nuclear shaping in cephalopod. PMID:21187923

Structure and expression of the Xenopus retinoblastoma gene.

PubMed

Destrée, O H; Lam, K T; Peterson-Maduro, L J; Eizema, K; Diller, L; Gryka, M A; Frebourg, T; Shibuya, E; Friend, S H

1992-09-01

We have cloned a Xenopus homology (XRb1) of the human retinoblastoma susceptibility gene. DNA sequence analysis shows that the XRb1 gene product is highly conserved in many regions. The leucine repeat motif and many of the potential cdc2 phosphorylation sites, as well as potential sites for other kinases, are retained. The region of the protein homologous to the SV40 T antigen binding site and the basic region directly C-terminal to the E1A binding site are all conserved. XRb1 gene expression at the RNA level was studied by Northern blot analysis. Transcripts of 4.2 and 10-kb are present as maternal RNA stores in the oocyte. While the 4.2-kb product is stable until at least the mid-blastula stage, the 10-kb transcript is selectively degraded. Between stages 11 and 13 the 10-kb transcript reappears and also a minor product of approximately 11 kb becomes apparent. Both the 4.2- and the 10-kb transcripts remain present until later stages of development and are also present in all adult tissues examined, although at differing levels. Antibodies raised against human p105Rb which recognize the protein product of the XRb1 gene, pXRb1, detect the Xenopus 99-kDa protein prior to the mid-blastula stage, but at lower levels than at later stages in development.

The nervus terminalis in larval and adult Xenopus laevis.

PubMed

Hofmann, M H; Meyer, D L

1989-09-25

Nervus terminalis (nt) projections were studied by HRP injections into one nostril in adult Xenopus and in Xenopus tadpoles. Central nt targets are: medial septum, preoptic nucleus, nucleus of the anterior commissure, and hypothalamus (mainly ipsilaterally). In Xenopus tadpoles, additional fibers reach the ipsilateral dorsal thalamus and the mesencephalic tegmentum, bilaterally; furthermore, hypothalamic projections are bilateral. Xenopus tadpole nt connections resemble those of adult urodeles more closely than the projections of frogs. However, Xenopus tadpoles lack nt innervation of the medial septum.

Synthesis of Lectin-Like Protein in Developing Cotyledons of Normal and Phytohemagglutinin-Deficient Phaseolus vulgaris.

PubMed

Vitale, A; Zoppè, M; Fabbrini, M S; Genga, A; Rivas, L; Bollini, R

1989-07-01

The genome of the common bean Phaseolus vulgaris contains a small gene family that encodes lectin and lectin-like proteins (phytohemagglutinin, arcelin, and others). One of these phytohemagglutinin-like genes was cloned by L. M. Hoffman et al. ([1982] Nucleic Acids Res 10: 7819-7828), but its product in bean cells has never been identified. We identified the product of this gene, referred to as lectin-like protein (LLP), as an abundant polypeptide synthesized on the endoplasmic reticulum (ER) of developing bean cotyledons. The gene product was first identified in extracts of Xenopus oocytes injected with either cotyledonary bean RNA or LLP-mRNA obtained by hybrid-selection with an LLP cDNA clone. A tryptic map of this protein was identical with a tryptic map of a polypeptide with the same SDS-PAGE mobility detectable in the ER of bean cotyledons pulse-labeled with either [(3)H]glucosamine or [(3)H]amino acids, both in a normal and in a phytohemagglutinin-deficient cultivar (cultivars Greensleeves and Pinto UI 111). Greensleeves LLP has M(r) 40,000 and most probably has four asparagine-linked glycans. Pinto UI 111 LLP has M(r) 38,500. Unlike phytohemagglutinin which is a tetramer, LLP appears to be a monomer by gel filtration analysis. Incorporation of [(3)H]amino acids indicates that synthesis of LLP accounts for about 3% of the proteins synthesized on the ER, a level similar to that of phytohemagglutinin.

Optimized S-Trityl-l-cysteine-Based Inhibitors of Kinesin Spindle Protein with Potent in Vivo Antitumor Activity in Lung Cancer Xenograft Models

PubMed Central

2013-01-01

The mitotic kinesin Eg5 is critical for the assembly of the mitotic spindle and is a promising chemotherapy target. Previously, we identified S-trityl-l-cysteine as a selective inhibitor of Eg5 and developed triphenylbutanamine analogues with improved potency, favorable drug-like properties, but moderate in vivo activity. We report here their further optimization to produce extremely potent inhibitors of Eg5 (Kiapp < 10 nM) with broad-spectrum activity against cancer cell lines comparable to the Phase II drug candidates ispinesib and SB-743921. They have good oral bioavailability and pharmacokinetics and induced complete tumor regression in nude mice explanted with lung cancer patient xenografts. Furthermore, they display fewer liabilities with CYP-metabolizing enzymes and hERG compared with ispinesib and SB-743921, which is important given the likely application of Eg5 inhibitors in combination therapies. We present the case for this preclinical series to be investigated in single and combination chemotherapies, especially targeting hematological malignancies. PMID:23394180

Building the Future: Post-transcriptional Regulation of Cell Fate Decisions Prior to the Xenopus Midblastula Transition.

PubMed

Sheets, Michael D

2015-01-01

In all animals, a critical period in early development is when embryonic cells switch from relying solely upon maternally deposited RNAs and proteins to relying upon molecules encoded by the zygotic genome. Xenopus embryos have served as a model for examining this switch, as well as the maternally controlled stages that prepare for it. In Xenopus, the robust activation of zygotic transcription occurs at the 12th cleavage division and is referred to as the midblastula transition (MBT). Prior to MBT, gene expression is regulated by post-transcriptional events including mRNA and protein localization, protein post-translational modification, and mRNA translation. After the MBT, appropriate transcriptional regulation of the zygotic genome becomes critical and predominates. However, it is important to realize that the first key cell fate decisions that have profound impacts on development occur prior to the MBT and these are governed by regulating the expression of maternally deposited regulatory mRNAs and proteins. In this chapter, I will discuss post-transcriptional mechanisms that function during the maternal stages of Xenopus development with an emphasis on mechanisms known to directly modulate cell fate decisions. Emerging approaches and technologies that will help better understand this phase of development will also be discussed. © 2015 Elsevier Inc. All rights reserved.

Ribosomal protein NtRPL17 interacts with kinesin-12 family protein NtKRP and functions in the regulation of embryo/seed size and radicle growth.

PubMed

Tian, Shujuan; Wu, Jingjing; Liu, Yuan; Huang, Xiaorong; Li, Fen; Wang, Zhaodan; Sun, Meng-Xiang

2017-11-28

We previously reported that a novel motor protein belonging to the kinesin-12 family, NtKRP, displays critical roles in regulating embryo and seed size establishment. However, it remains unknown exactly how NtKRP contributes to this developmental process. Here, we report that a 60S ribosomal protein NtRPL17 directly interacts with NtKRP. The phenotypes of NtRPL17 RNAi lines show notable embryo and seed size reduction. Structural observations of the NtRPL17-silenced embryos/seeds reveal that the embryo size reduction is due to a decrease in cell number. In these embryos, cell division cycle progression is delayed at the G2/M transition. These phenotypes are similar to that in NtKRP-silenced embryos/seeds, indicating that NtKRP and NtRPL17 function as partners in the same regulatory pathway during seed development and specifically regulate cell cycle progression to control embryo/seed size. This work reveals that NtRPL17, as a widely distributed ribosomal protein, plays a critical role in seed development and provides a new clue in the regulation of seed size. Confirmation of the interaction between NtKRP and NtRPL17 and their co-function in the control of the cell cycle also suggests that the mechanism might be conserved in both plants and animals. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Dynamic expression of the LAP family of genes during early development of Xenopus tropicalis.

PubMed

Yang, Qiutan; Lv, Xiaoyan; Kong, Qinghua; Li, Chaocui; Zhou, Qin; Mao, Bingyu

2011-10-01

The leucine-rich repeats and PDZ (LAP) family of genes are crucial for the maintenance of cell polarity as well as for epithelial homeostasis and tumor suppression in both vertebrates and invertebrates. Four members of this gene family are known: densin, erbin, scribble and lano. Here, we identified the four members of the LAP gene family in Xenopus tropicalis and studied their expression patterns during embryonic development. The Xenopus LAP proteins show a conserved domain structure that is similar to their homologs in other vertebrates. In Xenopus embryos, these genes were detected in animal cap cells at the early gastrula stage. At later stages of development, they were widely expressed in epithelial tissues that are highly polar in nature, including the neural epithelia, optic and otic vesicles, and in the pronephros. These data suggest that the roles of the Xenopus LAP genes in the control of cell polarity and morphogenesis are conserved during early development. Erbin and lano show similar expression patterns in the developing head, suggesting potential functional interactions between the two molecules in vivo.

Tumor immunology viewed from alternative animal models—the Xenopus story

PubMed Central

Banach, Maureen; Robert, Jacques

2017-01-01

a) Purpose of review Nonmammalian comparative animal models are important not only to gain fundamental evolutionary understanding of the complex interactions of tumors with the immune system, but also to better predict the applicability of novel immunotherapeutic approaches to humans. After reviewing recent advances in developing alternative models, we focus on the amphibian Xenopus laevis and its usefulness in deciphering the perplexing roles of MHC class I-like molecules and innate (i)T cells in tumor immunity. b) Recent findings Experiments using MHC-defined inbred and cloned animals, tumor cell lines, effective reagents, sequenced genomes, and adapted gene editing techniques in Xenopus, have revealed that the critical involvement of class I-like molecules and iT cells in tumor immunity has been conserved during evolution. c) Summary Comparative studies with the X. laevis tumor immunity model can contribute to the development of better and more efficient cancer immunotherapies. PMID:28944105

Reconstitution of dynein transport to the microtubule plus end by kinesin

PubMed Central

Roberts, Anthony J; Goodman, Brian S; Reck-Peterson, Samara L

2014-01-01

Cytoplasmic dynein powers intracellular movement of cargo toward the microtubule minus end. The first step in a variety of dynein transport events is the targeting of dynein to the dynamic microtubule plus end, but the molecular mechanism underlying this spatial regulation is not understood. Here, we reconstitute dynein plus-end transport using purified proteins from S. cerevisiae and dissect the mechanism using single-molecule microscopy. We find that two proteins–homologs of Lis1 and Clip170–are sufficient to couple dynein to Kip2, a plus-end-directed kinesin. Dynein is transported to the plus end by Kip2, but is not a passive passenger, resisting its own plus-end-directed motion. Two microtubule-associated proteins, homologs of Clip170 and EB1, act as processivity factors for Kip2, helping it overcome dynein's intrinsic minus-end-directed motility. This reveals how a minimal system of proteins transports a molecular motor to the start of its track. DOI: http://dx.doi.org/10.7554/eLife.02641.001 PMID:24916158

Hedgehog regulation of superficial slow muscle fibres in Xenopus and the evolution of tetrapod trunk myogenesis.

PubMed

Grimaldi, Annalisa; Tettamanti, Gianluca; Martin, Benjamin L; Gaffield, William; Pownall, Mary E; Hughes, Simon M

2004-07-01

In tetrapod phylogeny, the dramatic modifications of the trunk have received less attention than the more obvious evolution of limbs. In somites, several waves of muscle precursors are induced by signals from nearby tissues. In both amniotes and fish, the earliest myogenesis requires secreted signals from the ventral midline carried by Hedgehog (Hh) proteins. To determine if this similarity represents evolutionary homology, we have examined myogenesis in Xenopus laevis, the major species from which insight into vertebrate mesoderm patterning has been derived. Xenopus embryos form two distinct kinds of muscle cells analogous to the superficial slow and medial fast muscle fibres of zebrafish. As in zebrafish, Hh signalling is required for XMyf5 expression and generation of a first wave of early superficial slow muscle fibres in tail somites. Thus, Hh-dependent adaxial myogenesis is the likely ancestral condition of teleosts, amphibia and amniotes. Our evidence suggests that midline-derived cells migrate to the lateral somite surface and generate superficial slow muscle. This cell re-orientation contributes to the apparent rotation of Xenopus somites. Xenopus myogenesis in the trunk differs from that in the tail. In the trunk, the first wave of superficial slow fibres is missing, suggesting that significant adaptation of the ancestral myogenic programme occurred during tetrapod trunk evolution. Although notochord is required for early medial XMyf5 expression, Hh signalling fails to drive these cells to slow myogenesis. Later, both trunk and tail somites develop a second wave of Hh-independent slow fibres. These fibres probably derive from an outer cell layer expressing the myogenic determination genes XMyf5, XMyoD and Pax3 in a pattern reminiscent of amniote dermomyotome. Thus, Xenopus somites have characteristics in common with both fish and amniotes that shed light on the evolution of somite differentiation. We propose a model for the evolutionary adaptation of

The Elav-like protein HuR exerts translational control of viral internal ribosome entry sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rivas-Aravena, Andrea; Ramdohr, Pablo; Vallejos, Maricarmen

2009-09-30

The human embryonic-lethal abnormal vision (ELAV)-like protein, HuR, has been recently found to be involved in the regulation of protein synthesis. In this study we show that HuR participates in the translational control of the HIV-1 and HCV IRES elements. HuR functions as a repressor of HIV-1 IRES activity and acts as an activator of the HCV IRES. The effect of HuR was evaluated in three independent experimental systems, rabbit reticulocyte lysate, HeLa cells, and Xenopus laevis oocytes, using both overexpression and knockdown approaches. Furthermore, results suggest that HuR mediated regulation of HIV-1 and HCV IRESes does not require directmore » binding of the protein to the RNA nor does it need the nuclear translocation of the IRES-containing RNAs. Finally, we show that HuR has a negative impact on post-integration steps of the HIV-1 replication cycle. Thus, our observations yield novel insights into the role of HuR in the post-transcriptional regulation of HCV and HIV-1 gene expression.« less

Effect of the microtubule-associated protein tau on dynamics of single-headed motor proteins KIF1A

NASA Astrophysics Data System (ADS)

Sparacino, J.; Farías, M. G.; Lamberti, P. W.

2014-02-01

Intracellular transport based on molecular motors and its regulation are crucial to the functioning of cells. Filamentary tracks of the cells are abundantly decorated with nonmotile microtubule-associated proteins, such as tau. Motivated by experiments on kinesin-tau interactions [Dixit et al., Science 319, 1086 (2008), 10.1126/science.1152993] we developed a stochastic model of interacting single-headed motor proteins KIF1A that also takes into account the interactions between motor proteins and tau molecules. Our model reproduces experimental observations and predicts significant effects of tau on bound time and run length which suggest an important role of tau in regulation of kinesin-based transport.

Rotation of the stalk/neck and one head in a new crystal structure of the kinesin motor protein, Ncd

PubMed Central

Yun, Mikyung; Bronner, C.Eric; Park, Cheon-Gil; Cha, Sun-Shin; Park, Hee-Won; Endow, Sharyn A.

2003-01-01

Molecular motors undergo conformational changes to produce force and move along cytoskeletal filaments. Structural changes have been detected in kinesin motors; however, further changes are expected because previous crystal structures are in the same or closely related conformations. We report here a 2.5 Å crystal structure of the minus-end kinesin, Ncd, with the coiled-coil stalk/neck and one head rotated by ∼75° relative to the other head. The two heads are asymmetrically positioned with respect to the stalk and show asymmetry of nucleotide state: one head is fully occupied, but the other is unstably bound to ADP. Unlike previous structures, our new atomic model can be fit into cryoelectron microscopy density maps of the motor attached to microtubules, where it appears to resemble a one-head-bound motor with the stalk rotated towards the minus end. Interactions between neck and motor core residues, observed in the head that moves with the stalk, are disrupted in the other head, permitting rotation of the stalk/neck. The rotation could represent a force-producing stroke that directs the motor to the minus end. PMID:14532111

Expression of LRRC8/VRAC Currents in Xenopus Oocytes: Advantages and Caveats.

PubMed

Gaitán-Peñas, Héctor; Pusch, Michael; Estévez, Raúl

2018-03-02

Volume-regulated anion channels (VRACs) play a role in controlling cell volume by opening upon cell swelling. Apart from controlling cell volume, their function is important in many other physiological processes, such as transport of metabolites or drugs, and extracellular signal transduction. VRACs are formed by heteromers of the pannexin homologous protein LRRC8A (also named Swell1) with other LRRC8 members (B, C, D, and E). LRRC8 proteins are difficult to study, since they are expressed in all cells of our body, and the channel stoichiometry can be changed by overexpression, resulting in non-functional heteromers. Two different strategies have been developed to overcome this issue: complementation by transient transfection of LRRC8 genome-edited cell lines, and reconstitution in lipid bilayers. Alternatively, we have used Xenopus oocytes as a simple system to study LRRC8 proteins. Here, we have reviewed all previous experiments that have been performed with VRAC and LRRC8 proteins in Xenopus oocytes. We also discuss future strategies that may be used to perform structure-function analysis of the VRAC in oocytes and other systems, in order to understand its role in controlling multiple physiological functions.

XMAP310: A Xenopus Rescue-promoting Factor Localized to the Mitotic Spindle

PubMed Central

Andersen, Søren S.L.; Karsenti, Eric

1997-01-01

To understand the role of microtubule-associated proteins (MAPs) in the regulation of microtubule (MT) dynamics we have characterized MAPs prepared from Xenopus laevis eggs (Andersen, S.S.L., B. Buendia, J.E. Domínguez, A. Sawyer, and E. Karsenti. 1994. J. Cell Biol. 127:1289–1299). Here we report on the purification and characterization of a 310-kD MAP (XMAP310) that localizes to the nucleus in interphase and to mitotic spindle MTs in mitosis. XMAP310 is present in eggs, oocytes, a Xenopus tissue culture cell line, testis, and brain. We have purified XMAP310 to homogeneity from egg extracts. The purified protein cross-links pure MTs. Analysis of the effect of this protein on MT dynamics by time-lapse video microscopy has shown that it increases the rescue frequency 5–10-fold and decreases the shrinkage rate twofold. It has no effect on the growth rate or the catastrophe frequency. Microsequencing data suggest that XMAP230 and XMAP310 are novel MAPs. Although the three Xenopus MAPs characterized so far, XMAP215 (Vasquez, R.J., D.L. Gard, and L. Cassimeris. 1994. J. Cell Biol. 127:985–993), XMAP230, and XMAP310 are localized to the mitotic spindle, they have distinct effects on MT dynamics. While XMAP215 promotes rapid MT growth, XMAP230 decreases the catastrophe frequency and XMAP310 increases the rescue frequency. This may have important implications for the regulation of MT dynamics during spindle morphogenesis and chromosome segregation. PMID:9362515

Maternal program of apoptosis activated shortly after midblastula transition by overexpression of S-adenosylmethionine decarboxylase in Xenopus early embryos.

PubMed

Shiokawa, K; Kai, M; Higo, T; Kaito, C; Yokoska, J; Yasuhiko, Y; Kajita, E; Nagano, M; Yamada, Y; Shibata, M; Muto, T; Shinga, J; Hara, H; Takayama, E; Fukamachi, H; Yaoita, Y; Igarashi, K

2000-06-01

When we studied polyamine metabolism in Xenopus embryos, we cloned the cDNA for Xenopus S-adenosylmethionine decarboxylase (SAMDC), which converts SAM (S-adenosylmethionine), the methyl donor, into decarboxylated SAM (dcSAM), the aminopropyl donor, and microinjected its in vitro transcribed mRNA into Xenopus fertilized eggs. We found here that the mRNA injection induces a SAM deficient state in early embryos due to over-function of the overexpressed SAMDC, which in turn induces inhibition of protein synthesis. Such embryos developed quite normally until blastula stage, but stopped development at the early gastrula stage, due to induction of massive cell dissociation and cell autolysis, irrespective of the dosage and stage of the mRNA injection. We found that the dissociated cells were TUNEL-positive, contained fragmented nuclei with ladder-forming DNA, and furthermore, rescued completely by coinjection of Bcl-2 mRNA. Thus, overexpression of SAMDC in Xenopus embryos appeared to switch on apoptotic program, probably via inhibition of protein synthesis. Here, we briefly review our results together with those reported from other laboratories. After discussing the general importance of this newly discovered apoptotic program, we propose that the maternal program of apoptosis serves as a surveillance mechanism to eliminate metabolically severely-damaged cells and functions as a 'fail-safe' mechanism for normal development in Xenopus embryos.

Kinesin Mutations Cause Motor Neuron Disease Phenotypes by Disrupting Fast Axonal Transport in Drosophila

PubMed Central

Hurd, D. D.; Saxton, W. M.

1996-01-01

Previous work has shown that mutation of the gene that encodes the microtubule motor subunit kinesin heavy chain (Khc) in Drosophila inhibits neuronal sodium channel activity, action potentials and neurotransmitter secretion. These physiological defects cause progressive distal paralysis in larvae. To identify the cellular defects that cause these phenotypes, larval nerves were studied by light and electron microscopy. The axons of Khc mutants develop dramatic focal swellings along their lengths. The swellings are packed with fast axonal transport cargoes including vesicles, synaptic membrane proteins, mitochondria and prelysosomal organelles, but not with slow axonal transport cargoes such as cytoskeletal elements. Khc mutations also impair the development of larval motor axon terminals, causing dystrophic morphology and marked reductions in synaptic bouton numbers. These observations suggest that as the concentration of maternally provided wild-type KHC decreases, axonal organelles transported by kinesin periodically stall. This causes organelle jams that disrupt retrograde as well as anterograde fast axonal transport, leading to defective action potentials, dystrophic terminals, reduced transmitter secretion and progressive distal paralysis. These phenotypes parallel the pathologies of some vertebrate motor neuron diseases, including some forms of amyotrophic lateral sclerosis (ALS), and suggest that impaired fast axonal transport is a key element in those diseases. PMID:8913751

Detectable states, cycle fluxes, and motility scaling of molecular motor kinesin: An integrative kinetic graph theory analysis

NASA Astrophysics Data System (ADS)

Ren, Jie

2017-12-01

The process by which a kinesin motor couples its ATPase activity with concerted mechanical hand-over-hand steps is a foremost topic of molecular motor physics. Two major routes toward elucidating kinesin mechanisms are the motility performance characterization of velocity and run length, and single-molecular state detection experiments. However, these two sets of experimental approaches are largely uncoupled to date. Here, we introduce an integrative motility state analysis based on a theorized kinetic graph theory for kinesin, which, on one hand, is validated by a wealth of accumulated motility data, and, on the other hand, allows for rigorous quantification of state occurrences and chemomechanical cycling probabilities. An interesting linear scaling for kinesin motility performance across species is discussed as well. An integrative kinetic graph theory analysis provides a powerful tool to bridge motility and state characterization experiments, so as to forge a unified effort for the elucidation of the working mechanisms of molecular motors.

Essential roles of LEM-domain protein MAN1 during organogenesis in Xenopus laevis and overlapping functions of emerin.

PubMed

Reil, Michael; Dabauvalle, Marie-Christine

2013-01-01

Mutations in nuclear envelope proteins are linked to an increasing number of human diseases, called envelopathies. Mutations in the inner nuclear membrane protein emerin lead to X-linked Emery-Dreifuss muscular dystrophy, characterized by muscle weakness or wasting. Conversely, mutations in nuclear envelope protein MAN1 are linked to bone and skin disorders. Both proteins share a highly conserved domain, called LEM-domain. LEM proteins are known to interact with Barrier-to-autointegration factor and several transcription factors. Most envelopathies are tissue-specific, but knowledge on the physiological roles of related LEM proteins is still unclear. For this reason, we investigated the roles of MAN1 and emerin during Xenopus laevis organogenesis. Morpholino-mediated knockdown of MAN1 revealed that MAN1 is essential for the formation of eye, skeletal and cardiac muscle tissues. The MAN1 knockdown could be compensated by ectopic expression of emerin, leading to a proper organ development. Further investigations revealed that MAN1 is involved in regulation of genes essential for organ development and tissue homeostasis. Thereby our work supports that LEM proteins might be involved in signalling essential for organ development during early embryogenesis and suggests that loss of MAN1 may cause muscle and retina specific diseases. Copyright © 2013 Elsevier GmbH. All rights reserved.

Nearly 1000 Protein Identifications from 50 ng of Xenopus laevis Zygote Homogenate Using Online Sample Preparation on a Strong Cation Exchange Monolith Based Microreactor Coupled with Capillary Zone Electrophoresis.

PubMed

Zhang, Zhenbin; Sun, Liangliang; Zhu, Guijie; Cox, Olivia F; Huber, Paul W; Dovichi, Norman J

2016-01-05

A sulfonate-silica hybrid strong cation exchange monolith microreactor was synthesized and coupled to a linear polyacrylamide coated capillary for online sample preparation and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) bottom-up proteomic analysis. The protein sample was loaded onto the microreactor in an acidic buffer. After online reduction, alkylation, and digestion with trypsin, the digests were eluted with 200 mM ammonium bicarbonate at pH 8.2 for CZE-MS/MS analysis using 1 M acetic acid as the background electrolyte. This combination of basic elution and acidic background electrolytes results in both sample stacking and formation of a dynamic pH junction. 369 protein groups and 1274 peptides were identified from 50 ng of Xenopus laevis zygote homogenate, which is comparable with an offline sample preparation method, but the time required for sample preparation was decreased from over 24 h to less than 40 min. Dramatically improved performance was produced by coupling the reactor to a longer separation capillary (∼100 cm) and a Q Exactive HF mass spectrometer. 975 protein groups and 3749 peptides were identified from 50 ng of Xenopus protein using the online sample preparation method.

Different signaling pathway between sphingosine-1-phosphate and lysophosphatidic acid in Xenopus oocytes: functional coupling of the sphingosine-1-phosphate receptor to PLC-xbeta in Xenopus oocytes.

PubMed

Noh, S J; Kim, M J; Shim, S; Han, J K

1998-08-01

In Xenopus oocytes, both sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) activate Ca2+-dependent oscillatory Cl- currents by acting through membrane-bound receptors. External application of 50 microM S1P elicited a long-lasting oscillatory current that continued over 30 min from the beginning of oscillation, with 300 nA (n = 11) as a usual maximum peak of current, whereas 1-microM LPA treatment showed only transiently oscillating but more vigorous current responses, with 2,800 nA (n = 18) as a maximum peak amplitude. Both phospholipid-induced Ca2+-dependent Cl- currents were observed in the absence of extracellular Ca2+, were blocked by intracellular injection of the Ca2+ chelator, EGTA, and could not be elicited by treatment with thapsigargin, an inhibitor of endoplasmic reticulum (ER) Ca2+ ATPase. Intracellular Ca2+ release appeared to be from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store, because Cl- currents were blocked by heparin injection. Pretreatment with the aminosteroid, U-73122, an inhibitor of G protein-mediated phospholipase C (PLC) activation, to oocytes inhibited the current responses evoked both by S1P and LPA. However, when they were injected with 10 ng of antisense oligonucleotide (AS-ODN) against Xenopus phospholipase C (PLC-xbeta), oocytes could not respond to S1P application, whereas they responded normally to LPA, indicating that the S1P signaling pathway goes through PLC-xbeta, whereas LPA signaling goes through another unknown PLC. To determine the types of G proteins involved, we introduced AS-ODNs against four types of G-protein alpha subunits that were identified in Xenopus laevis; G(q)alpha, G11alpha, G0alpha, and G(i1)alpha. Among AS-ODNs against the G alphas tested, AS-G(q)alpha and AS-G(i1)alpha to S1P and AS-G(q)alpha and AS-G11alpha to LPA specifically reduced current responses, respectively, to about 20-30% of controls. These results demonstrate that LPA and S1P, although they have similar structural

Motility screen identifies Drosophila IGF-II mRNA-binding protein--zipcode-binding protein acting in oogenesis and synaptogenesis.

PubMed

Boylan, Kristin L M; Mische, Sarah; Li, Mingang; Marqués, Guillermo; Morin, Xavier; Chia, William; Hays, Thomas S

2008-02-01

The localization of specific mRNAs can establish local protein gradients that generate and control the development of cellular asymmetries. While all evidence underscores the importance of the cytoskeleton in the transport and localization of RNAs, we have limited knowledge of how these events are regulated. Using a visual screen for motile proteins in a collection of GFP protein trap lines, we identified the Drosophila IGF-II mRNA-binding protein (Imp), an ortholog of Xenopus Vg1 RNA binding protein and chicken zipcode-binding protein. In Drosophila, Imp is part of a large, RNase-sensitive complex that is enriched in two polarized cell types, the developing oocyte and the neuron. Using time-lapse confocal microscopy, we establish that both dynein and kinesin contribute to the transport of GFP-Imp particles, and that regulation of transport in egg chambers appears to differ from that in neurons. In Drosophila, loss-of-function Imp mutations are zygotic lethal, and mutants die late as pharate adults. Imp has a function in Drosophila oogenesis that is not essential, as well as functions that are essential during embryogenesis and later development. Germline clones of Imp mutations do not block maternal mRNA localization or oocyte development, but overexpression of a specific Imp isoform disrupts dorsal/ventral polarity. We report here that loss-of-function Imp mutations, as well as Imp overexpression, can alter synaptic terminal growth. Our data show that Imp is transported to the neuromuscular junction, where it may modulate the translation of mRNA targets. In oocytes, where Imp function is not essential, we implicate a specific Imp domain in the establishment of dorsoventral polarity.

Deletion of the Tail Domain of the Kinesin-5 Cin8 Affects Its Directionality*

PubMed Central

Düselder, André; Fridman, Vladimir; Thiede, Christina; Wiesbaum, Alice; Goldstein, Alina; Klopfenstein, Dieter R.; Zaitseva, Olga; Janson, Marcel E.; Gheber, Larisa; Schmidt, Christoph F.

2015-01-01

The bipolar kinesin-5 motors are one of the major players that govern mitotic spindle dynamics. Their bipolar structure enables them to cross-link and slide apart antiparallel microtubules (MTs) emanating from the opposing spindle poles. The budding yeast kinesin-5 Cin8 was shown to switch from fast minus-end- to slow plus-end-directed motility upon binding between antiparallel MTs. This unexpected finding revealed a new dimension of cellular control of transport, the mechanism of which is unknown. Here we have examined the role of the C-terminal tail domain of Cin8 in regulating directionality. We first constructed a stable dimeric Cin8/kinesin-1 chimera (Cin8Kin), consisting of head and neck linker of Cin8 fused to the stalk of kinesin-1. As a single dimeric motor, Cin8Kin switched frequently between plus and minus directionality along single MTs, demonstrating that the Cin8 head domains are inherently bidirectional, but control over directionality was lost. We next examined the activity of a tetrameric Cin8 lacking only the tail domains (Cin8Δtail). In contrast to wild-type Cin8, the motility of single molecules of Cin8Δtail in high ionic strength was slow and bidirectional, with almost no directionality switches. Cin8Δtail showed only a weak ability to cross-link MTs in vitro. In vivo, Cin8Δtail exhibited bias toward the plus-end of the MTs and was unable to support viability of cells as the sole kinesin-5 motor. We conclude that the tail of Cin8 is not necessary for bidirectional processive motion, but is controlling the switch between plus- and minus-end-directed motility. PMID:25991727

Crystal Structures of the Tetratricopeptide Repeat Domains of Kinesin Light Chains: Insight into Cargo Recognition Mechanisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Haizhong; Lee, Han Youl; Tong, Yufeng

Kinesin-1 transports various cargos along the axon by interacting with the cargos through its light chain subunit. Kinesin light chains (KLC) utilize its tetratricopeptide repeat (TPR) domain to interact with over 10 different cargos. Despite a high sequence identity between their TPR domains (87%), KLC1 and KLC2 isoforms exhibit differential binding properties towards some cargos. We determined the structures of human KLC1 and KLC2 tetratricopeptide repeat (TPR) domains using X-ray crystallography and investigated the different mechanisms by which KLCs interact with their cargos. Using isothermal titration calorimetry, we attributed the specific interaction between KLC1 and JNK-interacting protein 1 (JIP1) cargomore » to residue N343 in the fourth TRP repeat. Structurally, the N343 residue is adjacent to other asparagines and lysines, creating a positively charged polar patch within the groove of the TPR domain. Whereas, KLC2 with the corresponding residue S328 did not interact with JIP1. Based on these finding, we propose that N343 of KLC1 can form 'a carboxylate clamp' with its neighboring asparagine to interact with JIP1, similar to that of HSP70/HSP90 organizing protein-1's (HOP1) interaction with heat shock proteins. For the binding of cargos shared by KLC1 and KLC2, we propose a different site located within the groove but not involving N343. We further propose a third binding site on KLC1 which involves a stretch of polar residues along the inter-TPR loops that may form a network of hydrogen bonds to JIP3 and JIP4. Together, these results provide structural insights into possible mechanisms of interaction between KLC TPR domains and various cargo proteins.« less

Requirement of Xmsx-1 in the BMP-triggered ventralization of Xenopus embryos.

PubMed

Yamamoto, T S; Takagi, C; Ueno, N

2000-03-01

Signaling triggered by polypeptide growth factors leads to the activation of their target genes. Several homeobox genes are known to be induced in response to polypeptide growth factors in early Xenopus development. In particular, Xmsx-1, an amphibian homologue of vertebrate Msx-1, is well characterized as a target gene of bone morphogenetic protein (BMP). Here, using a dominant-negative form of Xmsx-1 (VP-Xmsx-1), which is a fusion protein made with the virus-derived VP16 activation domain, we have examined whether Xmsx-1 activity is required in the endogenous ventralizing pathway. VP-Xmsx-1 induced a secondary body axis, complete with muscle and neural tissues, when overexpressed in ventral blastomeres, suggesting that Xmsx-1 activity is necessary for both mesoderm and ectoderm to be ventralized. We have also examined the epistatic relationship between Xmsx-1 and another ventralizing homeobox protein, Xvent-1, and show that Xmsx-1 is likely to be acting upstream of Xvent-1. We propose that Xmsx-1 is required in the BMP-stimulated ventralization pathway that involves the downstream activation of Xvent-1.

Examining kinesin processivity within a general gating framework

PubMed Central

Andreasson, Johan OL; Milic, Bojan; Chen, Geng-Yuan; Guydosh, Nicholas R; Hancock, William O; Block, Steven M

2015-01-01

Kinesin-1 is a dimeric motor that transports cargo along microtubules, taking 8.2-nm steps in a hand-over-hand fashion. The ATP hydrolysis cycles of its two heads are maintained out of phase by a series of gating mechanisms, which lead to processive runs averaging ∼1 μm. A key structural element for inter-head coordination is the neck linker (NL), which connects the heads to the stalk. To examine the role of the NL in regulating stepping, we investigated NL mutants of various lengths using single-molecule optical trapping and bulk fluorescence approaches in the context of a general framework for gating. Our results show that, although inter-head tension enhances motor velocity, it is crucial neither for inter-head coordination nor for rapid rear-head release. Furthermore, cysteine-light mutants do not produce wild-type motility under load. We conclude that kinesin-1 is primarily front-head gated, and that NL length is tuned to enhance unidirectional processivity and velocity. DOI: http://dx.doi.org/10.7554/eLife.07403.001 PMID:25902401

The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs.

PubMed Central

Nebreda, A R; Hunt, T

1993-01-01

During studies of the activation and inactivation of the cyclin B-p34cdc2 protein kinase (MPF) in cell-free extracts of Xenopus oocytes and eggs, we found that a bacterially expressed fusion protein between the Escherichia coli maltose-binding protein and the Xenopus c-mos protein kinase (malE-mos) activated a 42 kDa MAP kinase. The activation of MAP kinase on addition of malE-mos was consistent, whereas the activation of MPF was variable and failed to occur in some oocyte extracts in which cyclin A or okadaic acid activated both MPF and MAP kinase. In cases when MPF activation was transient, MAP kinase activity declined after MPF activity was lost, and MAP kinase, but not MPF, could be maintained at a high level by the presence of malE-mos. When intact oocytes were treated with progesterone, however, the activation of MPF and MAP kinase occurred simultaneously, in contrast to the behaviour of extracts. These observations suggest that one role of c-mos may be to maintain high MAP kinase activity in meiosis. They also imply that the activation of MPF and MAP kinase in vivo are synchronous events that normally rely on an agent that has still to be identified. Images PMID:8387916

Structure of the SANT domain from the Xenopus chromatin remodeling factor ISWI

DOE Office of Scientific and Technical Information (OSTI.GOV)

Horton, John R.; Elgar, Stuart J.; Khan, Seema I.

2008-09-17

The SANT (Swi3, Ada2, N-Cor, and TFIIIB) module was first described as a putative DNA-binding domain with strong similarity to the helix-turn-helix DNA binding domain of Myb-related proteins. The X-ray structure of the C-terminal one third portion of the ATPase ISWI of Drosophila melangoaster, containing both SANT and SLIDE (SANT-Like ISWI Domain), confirmed the overall helix-turn-helix structural architecture of SANT as well as SLIDE. However, the DNA-contacting residues in Myb are not conserved in SANT and the structurally corresponding residues in the ISWI SANT domain are acidic, and therefore incompatible with DNA interaction. Recent studies suggested that SANT domains mightmore » be a histone-tail-binding module, including the DNA binding SANT domain of c-Myb. Here they present the X-ray structure of Xenopus laevis ISWI SANT domain, derived from limited proteolysis of a C-terminal fragment of ISWI protein.« less

Xenopus Bicaudal-C Is Required for the Differentiation of the Amphibian Pronephros

PubMed Central

Tran, Uyen; Mary Pickney, L.; Duygu Özpolat, B.; Wessely, Oliver

2007-01-01

The RNA-binding molecule Bicaudal-C regulates embryonic development in Drosophila and Xenopus. Interestingly, mouse mutants of Bicaudal-C do not show early patterning defects, but instead develop polycystic kidney disease (PKD). To further investigate the molecular mechanism of Bicaudal-C in kidney development, we analyzed its function in the developing amphibian pronephros. Bicaudal-C mRNA was present in the epithelial structures of the Xenopus pronephros, the tubules and the duct, but not the glomus. Inhibition of the translation of endogenous Bicaudal-C with antisense morpholino oligomers (xBic-C-MO) led to a PKD-like phenotype in Xenopus. Embryos lacking Bicaudal-C developed generalized edemas and dilated pronephric tubules and ducts. This phenotype was caused by impaired differentiation of the pronephros. Molecular markers specifically expressed in the late distal tubule were absent in xBic-C-MO-injected embryos. Furthermore, Bicaudal-C was not required for primary cilia formation, an important organelle affected in PKD. These data support the idea that Bicaudal-C functions downstream or parallel of a cilia-regulated signaling pathway. This pathway is required for terminal differentiation of the late distal tubule of the Xenopus pronephros and regulates renal epithelial cell differentiation, which - when disrupted - results in PKD. PMID:17521625

Xenopus-FV3 host-pathogen interactions and immune evasion.

PubMed

Jacques, Robert; Edholm, Eva-Stina; Jazz, Sanchez; Odalys, Torres-Luquis; Francisco, De Jesús Andino

2017-11-01

We first review fundamental insights into anti-ranavirus immunity learned with the Xenopus laevis/ranavirus FV3 model that are generally applicable to ectothermic vertebrates. We then further investigate FV3 genes involved in immune evasion. Focusing on FV3 knockout (KO) mutants defective for a putative viral caspase activation and recruitment domain-containing (CARD)-like protein (Δ64R-FV3), a β-hydroxysteroid dehydrogenase homolog (Δ52L-FV3), and an immediate-early18kDa protein (FV3-Δ18K), we assessed the involvement of these viral genes in replication, dissemination and interaction with peritoneal macrophages in tadpole and adult frogs. Our results substantiate the role of 64R and 52L as critical immune evasion genes, promoting persistence and dissemination in the host by counteracting type III IFN in tadpoles and type I IFN in adult frogs. Comparably, the substantial accumulation of genome copy numbers and exacerbation of type I and III IFN gene expression responses but deficient release of infectious virus suggests that 18K is a viral regulatory gene. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of functional neurotransmitter receptors in Xenopus oocytes after injection of human brain membranes

NASA Astrophysics Data System (ADS)

Miledi, Ricardo; Eusebi, Fabrizio; Martínez-Torres, Ataúlfo; Palma, Eleonora; Trettel, Flavia

2002-10-01

The Xenopus oocyte is a very powerful tool for studies of the structure and function of membrane proteins, e.g., messenger RNA extracted from the brain and injected into oocytes leads to the synthesis and membrane incorporation of many types of functional receptors and ion channels, and membrane vesicles from Torpedo electroplaques injected into oocytes fuse with the oocyte membrane and cause the appearance of functional Torpedo acetylcholine receptors and Cl channels. This approach was developed further to transplant already assembled neurotransmitter receptors from human brain cells to the plasma membrane of Xenopus oocytes. Membranes isolated from the temporal neocortex of a patient, operated for intractable epilepsy, were injected into oocytes and, within a few hours, the oocyte membrane acquired functional neurotransmitter receptors to -aminobutyric acid, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, and glycine. These receptors were also expressed in the plasma membrane of oocytes injected with mRNA extracted from the temporal neocortex of the same patient. All of this makes the Xenopus oocyte a more useful model than it already is for studies of the structure and function of many human membrane proteins and opens the way to novel pathophysiological investigations of some human brain disorders.

A self-powered kinesin-microtubule system for smart cargo delivery

NASA Astrophysics Data System (ADS)

Jia, Yi; Dong, Weiguang; Feng, Xiyun; Li, Jieling; Li, Junbai

2014-11-01

A smart self-powered cargo delivery system that is composed of creatine phosphate kinase (CPK) microspheres, kinesins and microtubules is demonstrated. The CPK microsphere not only acts as an ATP generation and buffering system, but also as a carrier for cargo transport, thus realizing the easy loading and self-powered delivery of cargos at the same time.A smart self-powered cargo delivery system that is composed of creatine phosphate kinase (CPK) microspheres, kinesins and microtubules is demonstrated. The CPK microsphere not only acts as an ATP generation and buffering system, but also as a carrier for cargo transport, thus realizing the easy loading and self-powered delivery of cargos at the same time. Electronic supplementary information (ESI) available: Experimental details, Fig. S1-S4, and Mov. S1-S6. See DOI: 10.1039/c4nr04454a

Interactive roles of Ras, insulin receptor substrate-1, and proteins with Src homology-2 domains in insulin signaling in Xenopus oocytes.

PubMed

Chuang, L M; Hausdorff, S F; Myers, M G; White, M F; Birnbaum, M J; Kahn, C R

1994-11-04

Insulin receptor substrate-1 (IRS-1) serves as the major immediate substrate of insulin/insulin-like growth factor (IGF)-1 receptors and following tyrosine phosphorylation binds to specific Src homology-2 (SH2) domain-containing proteins including the p85 subunit of phosphatidylinositol (PI) 3-kinase and GRB2, a molecule believed to link IRS-1 to the Ras pathway. To investigate how these SH2-containing signaling molecules interact to regulate insulin/IGF-1 action, IRS-1, glutathione S-transferase (GST)-SH2 domain fusion proteins and Ras proteins were microinjected into Xenopus oocytes. We found that pleiotropic insulin actions are mediated by IRS-1 through two independent, but convergent, pathways involving PI 3-kinase and GRB2. Thus, microinjection of GST-fusion proteins of either p85 or GRB2 inhibited IRS-1-dependent activation of mitogen-activated protein (MAP) and S6 kinases and oocyte maturation, although only the GST-SH2 of p85 reduced insulin-stimulated PI 3-kinase activation. Co-injection of a dominant negative Ras (S17N) with IRS-1 inhibited insulin-stimulated MAP and S6 kinase activation. Micro-injection of activated [Arg12,Thr59]Ras increased basal MAP and S6 kinase activities and sensitized the oocytes to insulin-stimulated maturation without altering insulin-stimulated PI 3-kinase. The Ras-enhanced oocyte maturation response, but not the elevated basal level of MAP and S6 kinase, was partially blocked by the SH2-p85, but not SH2-GRB2. These data strongly suggest that IRS-1 can mediate many of insulin's actions on cellular enzyme activation and cell cycle progression requires binding and activation of multiple different SH2-domain proteins.

Host-defense peptides from skin secretions of the octoploid frogs Xenopus vestitus and Xenopus wittei (Pipidae): insights into evolutionary relationships.

PubMed

Mechkarska, Milena; Coquet, Laurent; Leprince, Jérôme; Jouenne, Thierry; Vaudry, Hubert; Michalak, Katarzyna; Michalak, Pawel; Conlon, J Michael

2014-09-01

The primary structures of host-defense peptides have proved useful in elucidating the evolution history of frogs. Peptidomic analysis was used to compare the diversity of host-defense peptides in norepinephrine-stimulated skin secretions from the octoploid frogs, Xenopus vestitus (Kivu clawed frog) and Xenopus wittei (De Witte's clawed frog) in the family Pipidae. Structural characterization demonstrated that the X. vestitus peptides belong to the magainin (3 peptides), peptide glycine-leucine-amide (PGLa; 4 peptides), xenopsin-precursor fragment (XPF; 1 peptide), and caerulein-precursor fragment (CPF; 5 peptides) families. The X. wittei peptides comprise magainin (4 peptides), PGLa (1 peptide), XPF (2 peptides), and CPF (7 peptides). In addition, secretions from both species contain caerulein, identical to the peptide from Xenopus laevis, but X. wittei secretions contains the novel peptide [R4K]xenopsin. The variability in the numbers of paralogs in each peptide family indicates a selective silencing of the host-defense peptide genes following the polyploidization events. The primary structures of the peptides provide insight into phylogenetic relationships among the octoploid Xenopus frogs. The data support a sister-group relationship between X. vestitus and Xenopus lenduensis, suggestive of bifurcating speciation after allopolyploidization, whereas X. wittei is more closely related to the Xenopus amieti-Xenopus andrei group suggesting a common tetraploid ancestor. Consistent with previous data, the CPF peptides showed the highest growth inhibitory activity against bacteria with CPF-W6 (GIGSLLAKAAKLAAGLV.NH2) combining high antimicrobial potency against Staphylococcus aureus (MIC=4 μM) with relatively low hemolytic activity (LC50=190 μM). Copyright © 2014 Elsevier Inc. All rights reserved.

Xenopus extract approaches to studying microtubule organization and signaling in cytokinesis

PubMed Central

Field, Christine M.; Pelletier, James F.; Mitchison, Timothy J.

2017-01-01

We report optimized methods for preparing actin-intact Xenopus egg extract. This extract is minimally perturbed, undiluted egg cytoplasm where the cell cycle can be experimentally controlled. It contains abundant organelles and glycogen, and supports active metabolism and cytoskeletal dynamics that closely mimic egg physiology. The concentration of the most abundant ~11,000 proteins is known from mass spectrometry. Actin-intact egg extract can be used for analysis of actin dynamics and interaction of actin with other cytoplasmic systems, as well as microtubule organization. It can be spread as thin layers, and naturally depletes oxygen though mitochondrial metabolism, which makes it ideal for fluorescence imaging. When combined with artificial lipid bilayers, it allows reconstitution and analysis of the spatially controlled signaling that positions the cleavage furrow during early cytokinesis. Actin-intact extract is generally useful for probing the biochemistry and biophysics of the large Xenopus egg. Protocols are provided for preparation of actin-intact egg extract, control of the cell cycle, fluorescent probes for cytoskeleton and cytoskeleton-dependent signaling, preparation of glass surfaces for imaging experiments, and immunodepletion to probe the role of specific proteins and protein complexes. We also describe methods for adding supported lipid bilayers to mimic the plasma membrane and for confining in microfluidic droplets to explore size scaling issues. PMID:28065319

Evolution of Heat Sensors Drove Shifts in Thermosensation between Xenopus Species Adapted to Different Thermal Niches*

PubMed Central

Saito, Shigeru; Ohkita, Masashi; Saito, Claire T.; Takahashi, Kenji; Tominaga, Makoto; Ohta, Toshio

2016-01-01

Temperature is one of the most critical environmental factors affecting survival, and thus species that inhabit different thermal niches have evolved thermal sensitivities suitable for their respective habitats. During the process of shifting thermal niches, various types of genes expressed in diverse tissues, including those of the peripheral to central nervous systems, are potentially involved in the evolutionary changes in thermosensation. To elucidate the molecular mechanisms behind the evolution of thermosensation, thermal responses were compared between two species of clawed frogs (Xenopus laevis and Xenopus tropicalis) adapted to different thermal environments. X. laevis was much more sensitive to heat stimulation than X. tropicalis at the behavioral and neural levels. The activity and sensitivity of the heat-sensing TRPA1 channel were higher in X. laevis compared with those of X. tropicalis. The thermal responses of another heat-sensing channel, TRPV1, also differed between the two Xenopus species. The species differences in Xenopus TRPV1 heat responses were largely determined by three amino acid substitutions located in the first three ankyrin repeat domains, known to be involved in the regulation of rat TRPV1 activity. In addition, Xenopus TRPV1 exhibited drastic species differences in sensitivity to capsaicin, contained in chili peppers, between the two Xenopus species. Another single amino acid substitution within Xenopus TRPV1 is responsible for this species difference, which likely alters the neural and behavioral responses to capsaicin. These combined subtle amino acid substitutions in peripheral thermal sensors potentially serve as a driving force for the evolution of thermal and chemical sensation. PMID:27022021

Amphibian (Xenopus sp.) iodothyronine deiodinase ...

EPA Pesticide Factsheets

The U.S. EPA-MED amphibian thyroid group is currently screening chemicals for inhibition of human iodothyronine deiodinase activity as components of the thyroid system important in human development. Amphibians are a bellwether taxonomic group to gauge toxicity of chemicals in the environment. Amphibian thyroid function is not only important in development but also metamorphosis. Xenopus sp. have been used extensively as model organisms and are well characterized genetically. We propose to screen a list of chemicals (selected from the human DIO screening results) to test for inhibition of Xenopus deiodinases. Large quantities of the enzymes will be produced using an adenovirus system. Our preliminary results show that there may be catalytic differences between human and Xenopus deiodinases. The Twin Ports Early Career Scientists is a new group formed within the Duluth-Superior scientific community. This presentation will provide a basic introduction to my research and our mission at EPA, and help to establish networking and collaboration relationships across disciplines and institutions.

Morphology of the caudal spinal cord in Rana (Ranidae) and Xenopus (Pipidae) tadpoles.

PubMed

Nishikawa, K; Wassersug, R

1988-03-08

Using a variety of neuroanatomical and histological techniques, we compare the spinal cord and peripheral nerve distribution in the tails of larvae from Xenopus laevis and three species of Rana. The relatively large, postsacral spinal cord of Xenopus contains abundant motoneurons and their axons. Spinal nerves exit from the spinal cord in a regular array, one nerve per myotome, from the cervical region to near the end of the tail. Somata of motoneurons innervating caudal myotomes are found along the entire length of the tail. In contrast, the caudal cord of Rana is reduced to a filum terminale consisting of little more than an ependymal tube; spinal nerves to all caudal myotomes leave the cord in the sacral region and reach their motor targets via a cauda equina and caudal plexus. Motoneuron cell bodies innervating caudal myotomes are found only in the sacral region. The Rana larval pattern is similar to that of adult frogs and mammals, whereas the Xenopus larval pattern is more like that of salamanders and reptiles. These gross neuroanatomical differences are not due to differences in the size or developmental stage of the tadpoles, but instead are associated with differences in the swimming behavior of the larvae. The presence of motoneurons in the caudal spinal cord of Xenopus may provide local intermyotomal control within the tail; the elongated topography of the cord appears to permit finer, rostral-to-caudal regulation of neuromuscular activity. The Rana spinal cord, on the other hand--with motoneurons clustered anteriorly--may produce concurrent firing of adjacent ipsilateral myotomes, but at the expense of fine intermyotomal regulation. The fact that nerves in the tail of Xenopus enter and exit from the spinal cord locally, as opposed to far anteriorly as in Rana, means that for tadpoles of the same size, reflex arc lengths are many times shorter in Xenopus.

Small Molecule Suppressors of Drosophila Kinesin Deficiency Rescue Motor Axon Development in a Zebrafish Model of Spinal Muscular Atrophy

PubMed Central

Gassman, Andrew; Hao, Le T.; Bhoite, Leena; Bradford, Chad L.; Chien, Chi-Bin; Beattie, Christine E.; Manfredi, John P.

2013-01-01

Proximal spinal muscular atrophy (SMA) is the most common inherited motor neuropathy and the leading hereditary cause of infant mortality. Currently there is no effective treatment for the disease, reflecting a need for pharmacologic interventions that restore performance of dysfunctional motor neurons or suppress the consequences of their dysfunction. In a series of assays relevant to motor neuron biology, we explored the activities of a collection of tetrahydroindoles that were reported to alter the metabolism of amyloid precursor protein (APP). In Drosophila larvae the compounds suppressed aberrant larval locomotion due to mutations in the Khc and Klc genes, which respectively encode the heavy and light chains of kinesin-1. A representative compound of this class also suppressed the appearance of axonal swellings (alternatively termed axonal spheroids or neuritic beads) in the segmental nerves of the kinesin-deficient Drosophila larvae. Given the importance of kinesin-dependent transport for extension and maintenance of axons and their growth cones, three members of the class were tested for neurotrophic effects on isolated rat spinal motor neurons. Each compound stimulated neurite outgrowth. In addition, consistent with SMA being an axonopathy of motor neurons, the three axonotrophic compounds rescued motor axon development in a zebrafish model of SMA. The results introduce a collection of small molecules as pharmacologic suppressors of SMA-associated phenotypes and nominate specific members of the collection for development as candidate SMA therapeutics. More generally, the results reinforce the perception of SMA as an axonopathy and suggest novel approaches to treating the disease. PMID:24023935

Genetic screens for mutations affecting development of Xenopus tropicalis.

PubMed

Goda, Tadahiro; Abu-Daya, Anita; Carruthers, Samantha; Clark, Matthew D; Stemple, Derek L; Zimmerman, Lyle B

2006-06-01

We present here the results of forward and reverse genetic screens for chemically-induced mutations in Xenopus tropicalis. In our forward genetic screen, we have uncovered 77 candidate phenotypes in diverse organogenesis and differentiation processes. Using a gynogenetic screen design, which minimizes time and husbandry space expenditures, we find that if a phenotype is detected in the gynogenetic F2 of a given F1 female twice, it is highly likely to be a heritable abnormality (29/29 cases). We have also demonstrated the feasibility of reverse genetic approaches for obtaining carriers of mutations in specific genes, and have directly determined an induced mutation rate by sequencing specific exons from a mutagenized population. The Xenopus system, with its well-understood embryology, fate map, and gain-of-function approaches, can now be coupled with efficient loss-of-function genetic strategies for vertebrate functional genomics and developmental genetics.

Optimization of gene delivery methods in Xenopus laevis kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of Xenopus inner ear genes

PubMed Central

Ramirez-Gordillo, Daniel; Trujillo-Provencio, Casilda; Knight, V. Bleu; Serrano, Elba E.

2014-01-01

The Xenopus inner ear provides a useful model for studies of hearing and balance because it shares features with the mammalian inner ear, and because amphibians are capable of regenerating damaged mechanosensory hair cells. The structure and function of many proteins necessary for inner ear function have yet to be elucidated and require methods for analysis. To this end, we seek to characterize Xenopus inner ear genes outside of the animal model through heterologous expression in cell lines. As part of this effort, we aimed to optimize physical (electroporation), chemical (lipid-mediated; Lipofectamine™ 2000, Metafectene® Pro), and biological (viral-mediated; BacMam virus Cellular Lights™ Tubulin-RFP) gene delivery methods in amphibian (Xenopus; A6) cells and mammalian (Chinese hamster ovary (CHO)) cells. We successfully introduced the commercially available pEGFP-N3, pmCherry-N1, pEYFP-Tubulin, and Cellular Lights™ Tubulin-RFP fluorescent constructs to cells and evaluated their transfection or transduction efficiencies using the three gene delivery methods. In addition, we analyzed the transfection efficiency of a novel construct synthesized in our laboratory by cloning the Xenopus inner ear calcium-activated potassium channel β1 subunit, then subcloning the subunit into the pmCherry-N1 vector. Every gene delivery method was significantly more effective in CHO cells. Although results for the A6 cell line were not statistically significant, both cell lines illustrate a trend towards more efficient gene delivery using viral-mediated methods; however the cost of viral transduction is also much higher. Our findings demonstrate the need to improve gene delivery methods for amphibian cells and underscore the necessity for a greater understanding of amphibian cell biology. PMID:21959846

The G-protein-coupled receptor, GPR84, is important for eye development in Xenopus laevis.

PubMed

Perry, Kimberly J; Johnson, Verity R; Malloch, Erica L; Fukui, Lisa; Wever, Jason; Thomas, Alvin G; Hamilton, Paul W; Henry, Jonathan J

2010-11-01

G-protein-coupled receptors (GPCRs) represent diverse, multifamily groups of cell signaling receptors involved in many cellular processes. We identified Xenopus laevis GPR84 as a member of the A18 subfamily of GPCRs. During development, GPR84 is detected in the embryonic lens placode, differentiating lens fiber cells, retina, and cornea. Anti-sense morpholino oligonucleotide-mediated knockdown and RNA rescue experiments demonstrate GPR84's importance in lens, cornea, and retinal development. Examination of cell proliferation using an antibody against histone H3 S10P reveals significant increases in the lens and retina following GPR84 knockdown. Additionally, there was also an increase in apoptosis in the retina and lens, as revealed by TUNEL assay. Reciprocal transplantation of the presumptive lens ectoderm between uninjected controls and morpholino-injected embryos demonstrates that GPR84 is necessary in the retina for proper development of the retina, as well as other eye tissues including the lens and cornea. © 2010 Wiley-Liss, Inc.

The G-protein-coupled receptor, GPR84, is important for eye development in Xenopus laevis

PubMed Central

Perry, Kimberly J.; Johnson, Verity R.; Malloch, Erica L.; Fukui, Lisa; Wever, Jason; Thomas, Alvin G.; Hamilton, Paul W.; Henry, Jonathan J.

2010-01-01

G-protein-coupled receptors (GPCRs) represent diverse, multifamily groups of cell signaling receptors involved in many cellular processes. We identified Xenopus laevis GPR84 as a member of the A18 subfamily of GPCRs. During development, GPR84 is detected in the embryonic lens placode, differentiating lens fiber cells, retina and cornea. Anti-sense morpholino oligonucleotide-mediated knockdown and RNA rescue experiments demonstrate GPR84’s importance in lens, cornea and retinal development. Examination of cell proliferation using an antibody against histone H3 S10P reveals significant increases in the lens and retina following GPR84 knockdown. Additionally, there was also an increase in apoptosis in the retina and lens, as revealed by TUNEL assay. Reciprocal transplantation of the presumptive lens ectoderm between uninjected controls and morpholino injected embryos demonstrates that GPR84 is necessary in the retina for proper development of the retina, as well as other eye tissues including the lens and cornea. PMID:20925114

GSK-3 regulates transport of kinesin-1 driven cargos in vivo

NASA Astrophysics Data System (ADS)

Leidel, Christina; Weaver, Carole; Szpankowski, Lukasz; Goldstein, Lawrence S. B.; Shubeita, George T.; CenterNonlinear Dynamics, Department of Physics, University of Texas At Austin Collaboration; Hhmi, Department of Cellular; Molecular Medicine, Univ. Of California Collaboration

2011-03-01

The Glycogen Synthase Kinase 3 (GSK-3) has been linked to many aspects of the development of Alzheimer's disease and was proposed to play a role in the transport of the Amyloid Precursor Protein (APP) by kinesin-1 motors. Using Drosophila embryos and larvae with altered GSK-3 expression, we characterize motor transport of cargos including APP and lipid droplets using DIC microscopy, high-resolution video tracking, fluorescence, and in vivo stall force measurements with optical tweezers. By comparing cargo velocities and run lengths we find that GSK-3 is a required negative regulator of in vivo transport. Stall force measurements on lipid droplets reveal that enhanced transport under conditions of reduced GSK-3 is a result of a larger number of active motors hauling the cargo. Our findings have implications on the use of GSK-3 inhibitors in treatment of Alzheimer's disease.

Predicting the stochastic guiding of kinesin-driven microtubules in microfabricated tracks: a statistical-mechanics-based modeling approach.

PubMed

Lin, Chih-Tin; Meyhofer, Edgar; Kurabayashi, Katsuo

2010-01-01

Directional control of microtubule shuttles via microfabricated tracks is key to the development of controlled nanoscale mass transport by kinesin motor molecules. Here we develop and test a model to quantitatively predict the stochastic behavior of microtubule guiding when they mechanically collide with the sidewalls of lithographically patterned tracks. By taking into account appropriate probability distributions of microscopic states of the microtubule system, the model allows us to theoretically analyze the roles of collision conditions and kinesin surface densities in determining how the motion of microtubule shuttles is controlled. In addition, we experimentally observe the statistics of microtubule collision events and compare our theoretical prediction with experimental data to validate our model. The model will direct the design of future hybrid nanotechnology devices that integrate nanoscale transport systems powered by kinesin-driven molecular shuttles.

Xenopus microRNA genes are predominantly located within introns and are differentially expressed in adult frog tissues via post-transcriptional regulation

PubMed Central

Tang, Guo-Qing; Maxwell, E. Stuart

2008-01-01

The amphibian Xenopus provides a model organism for investigating microRNA expression during vertebrate embryogenesis and development. Searching available Xenopus genome databases using known human pre-miRNAs as query sequences, more than 300 genes encoding 142 Xenopus tropicalis miRNAs were identified. Analysis of Xenopus tropicalis miRNA genes revealed a predominate positioning within introns of protein-coding and nonprotein-coding RNA Pol II-transcribed genes. MiRNA genes were also located in pre-mRNA exons and positioned intergenically between known protein-coding genes. Many miRNA species were found in multiple locations and in more than one genomic context. MiRNA genes were also clustered throughout the genome, indicating the potential for the cotranscription and coordinate expression of miRNAs located in a given cluster. Northern blot analysis confirmed the expression of many identified miRNAs in both X. tropicalis and X. laevis. Comparison of X. tropicalis and X. laevis blots revealed comparable expression profiles, although several miRNAs exhibited species-specific expression in different tissues. More detailed analysis revealed that for some miRNAs, the tissue-specific expression profile of the pri-miRNA precursor was distinctly different from that of the mature miRNA profile. Differential miRNA precursor processing in both the nucleus and cytoplasm was implicated in the observed tissue-specific differences. These observations indicated that post-transcriptional processing plays an important role in regulating miRNA expression in the amphibian Xenopus. PMID:18032731

Kinesin is the motor for microtubule-mediated Golgi-to-ER membrane traffic [published errata appear in J Cell Biol 1995 Mar;128(5):following 988 and 1995 May;129(3):893

PubMed Central

1995-01-01

The distribution and dynamics of both the ER and Golgi complex in animal cells are known to be dependent on microtubules; in many cell types the ER extends toward the plus ends of microtubules at the cell periphery and the Golgi clusters at the minus ends of microtubules near the centrosome. In this study we provide evidence that the microtubule motor, kinesin, is present on membranes cycling between the ER and Golgi and powers peripherally directed movements of membrane within this system. Immunolocalization of kinesin at both the light and electron microscopy levels in NRK cells using the H1 monoclonal antibody to kinesin heavy chain, revealed kinesin to be associated with all membranes of the ER/Golgi system. At steady-state at 37 degrees C, however, kinesin was most concentrated on peripherally distributed, pre- Golgi structures containing beta COP and vesicular stomatitis virus glycoprotein newly released from the ER. Upon temperature reduction or nocodazole treatment, kinesin's distribution shifted onto the Golgi, while with brefeldin A (BFA)-treatment, kinesin could be found in both Golgi-derived tubules and in the ER. This suggested that kinesin associates with membranes that constitutively cycle between the ER and Golgi. Kinesin's role on these membranes was examined by microinjecting kinesin antibody. Golgi-to-ER but not ER-to-Golgi membrane transport was found to be inhibited by the microinjected anti-kinesin, suggesting kinesin powers the microtubule plus end-directed recycling of membrane to the ER, and remains inactive on pre-Golgi intermediates that move toward the Golgi complex. PMID:7844144

A Statistical Physicist's Approach to Biological Motion: From the the Kinesin Walk to Muscle Contraction

NASA Astrophysics Data System (ADS)

Vicsek, Tamas

1997-03-01

It is demonstrated that a wide range of experimental results on biological motion can be successfully interpreted in terms of statistical physics motivated models taking into account the relevant microscopic details of motor proteins and allowing analytic solutions. Two important examples are considered, i) the motion of a single kinesin molecule along microtubules inside individual cells and ii) muscle contraction which is a macroscopic phenomenon due to the collective action of a large number of myosin heads along actin filaments. i) Recently individual two-headed kinesin molecules have been studied in in vitro motility assays revealing a number of their peculiar transport properties. Here we propose a simple and robust model for the kinesin stepping process with elastically coupled Brownian heads showing all of these properties. The analytic treatment of our model results in a very good fit to the experimental data and practically has no free parameters. ii) Myosin is an ATPase enzyme that converts the chemical energy stored in ATP molecules into mechanical work. During muscle contraction, the myosin cross-bridges attach to the actin filaments and exert force on them yielding a relative sliding of the actin and myosin filaments. In this paper we present a simple mechanochemical model for the cross-bridge interaction involving the relevant kinetic data and providing simple analytic solutions for the mechanical properties of muscle contraction, such as the force-velocity relationship or the relative number of the attached cross-bridges. So far the only analytic formula which could be fitted to the measured force-velocity curves has been the well known Hill equation containing parameters lacking clear microscopic origin. The main advantages of our new approach are that it explicitly connects the mechanical data with the kinetic data and the concentration of the ATP and ATPase products and as such it leads to new analytic solutions which agree extremely well with a

Engineering of a novel Ca{sup 2+}-regulated kinesin molecular motor using a calmodulin dimer linker

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shishido, Hideki; Maruta, Shinsaku, E-mail: maruta@soka.ac.jp

Highlights: Black-Right-Pointing-Pointer Engineered kinesin-M13 and calmodulin involving single cysteine were prepared. Black-Right-Pointing-Pointer CaM mutant was cross-linked to dimer by bifunctional thiol reactive reagent. Black-Right-Pointing-Pointer Kinesin-M13 was dimerized via CaM dimer in the presence of calcium. Black-Right-Pointing-Pointer Function of the engineered kinesin was regulated by a Ca{sup 2+}-calmodulin dimer linker. -- Abstract: The kinesin-microtubule system holds great promise as a molecular shuttle device within biochips. However, one current barrier is that such shuttles do not have 'on-off' control of their movement. Here we report the development of a novel molecular motor powered by an accelerator and brake system, using a kinesinmore » monomer and a calmodulin (CaM) dimer. The kinesin monomer, K355, was fused with a CaM target peptide (M13 peptide) at the C-terminal part of the neck region (K355-M13). We also prepared CaM dimers using CaM mutants (Q3C), (R86C), or (A147C) and crosslinkers that react with cysteine residues. Following induction of K355-M13 dimerization with CaM dimers, we measured K355-M13 motility and found that it can be reversibly regulated in a Ca{sup 2+}-dependent manner. We also found that velocities of K355-M13 varied depending on the type and crosslink position of the CaM dimer used; crosslink length also had a moderate effect on motility. These results suggest Ca{sup 2+}-dependent dimerization of K355-M13 could be used as a novel molecular shuttle, equipped with an accelerator and brake system, for biochip applications.« less

Identification of new regulators of embryonic patterning and morphogenesis in Xenopus gastrulae by RNA sequencing.

PubMed

Popov, Ivan K; Kwon, Taejoon; Crossman, David K; Crowley, Michael R; Wallingford, John B; Chang, Chenbei

2017-06-15

During early vertebrate embryogenesis, cell fate specification is often coupled with cell acquisition of specific adhesive, polar and/or motile behaviors. In Xenopus gastrulae, tissues fated to form different axial structures display distinct motility. The cells in the early organizer move collectively and directionally toward the animal pole and contribute to anterior mesendoderm, whereas the dorsal and the ventral-posterior trunk tissues surrounding the blastopore of mid-gastrula embryos undergo convergent extension and convergent thickening movements, respectively. While factors regulating cell lineage specification have been described in some detail, the molecular machinery that controls cell motility is not understood in depth. To gain insight into the gene battery that regulates both cell fates and motility in particular embryonic tissues, we performed RNA sequencing (RNA-seq) to investigate differentially expressed genes in the early organizer, the dorsal and the ventral marginal zone of Xenopus gastrulae. We uncovered many known signaling and transcription factors that have been reported to play roles in embryonic patterning during gastrulation. We also identified many uncharacterized genes as well as genes that encoded extracellular matrix (ECM) proteins or potential regulators of actin cytoskeleton. Co-expression of a selected subset of the differentially expressed genes with activin in animal caps revealed that they had distinct ability to block activin-induced animal cap elongation. Most of these factors did not interfere with mesodermal induction by activin, but an ECM protein, EFEMP2, inhibited activin signaling and acted downstream of the activated type I receptor. By focusing on a secreted protein kinase PKDCC1, we showed with overexpression and knockdown experiments that PKDCC1 regulated gastrulation movements as well as anterior neural patterning during early Xenopus development. Overall, our studies identify many differentially expressed

Parsing the roles of neck-linker docking and tethered head diffusion in the stepping dynamics of kinesin.

PubMed

Zhang, Zhechun; Goldtzvik, Yonathan; Thirumalai, D

2017-11-14

Kinesin walks processively on microtubules (MTs) in an asymmetric hand-over-hand manner consuming one ATP molecule per 16-nm step. The individual contributions due to docking of the approximately 13-residue neck linker to the leading head (deemed to be the power stroke) and diffusion of the trailing head (TH) that contributes in propelling the motor by 16 nm have not been quantified. We use molecular simulations by creating a coarse-grained model of the MT-kinesin complex, which reproduces the measured stall force as well as the force required to dislodge the motor head from the MT, to show that nearly three-quarters of the step occurs by bidirectional stochastic motion of the TH. However, docking of the neck linker to the leading head constrains the extent of diffusion and minimizes the probability that kinesin takes side steps, implying that both the events are necessary in the motility of kinesin and for the maintenance of processivity. Surprisingly, we find that during a single step, the TH stochastically hops multiple times between the geometrically accessible neighboring sites on the MT before forming a stable interaction with the target binding site with correct orientation between the motor head and the [Formula: see text] tubulin dimer.

RMND5 from Xenopus laevis is an E3 ubiquitin-ligase and functions in early embryonic forebrain development.

PubMed

Pfirrmann, Thorsten; Villavicencio-Lorini, Pablo; Subudhi, Abinash K; Menssen, Ruth; Wolf, Dieter H; Hollemann, Thomas

2015-01-01

In Saccharomyces cerevisiae the Gid-complex functions as an ubiquitin-ligase complex that regulates the metabolic switch between glycolysis and gluconeogenesis. In higher organisms six conserved Gid proteins form the CTLH protein-complex with unknown function. Here we show that Rmnd5, the Gid2 orthologue from Xenopus laevis, is an ubiquitin-ligase embedded in a high molecular weight complex. Expression of rmnd5 is strongest in neuronal ectoderm, prospective brain, eyes and ciliated cells of the skin and its suppression results in malformations of the fore- and midbrain. We therefore suggest that Xenopus laevis Rmnd5, as a subunit of the CTLH complex, is a ubiquitin-ligase targeting an unknown factor for polyubiquitination and subsequent proteasomal degradation for proper fore- and midbrain development.

Evolution of Courtship Songs in Xenopus : Vocal Pattern Generation and Sound Production.

PubMed

Leininger, Elizabeth C; Kelley, Darcy B

2015-01-01

The extant species of African clawed frogs (Xenopus and Silurana) provide an opportunity to link the evolution of vocal characters to changes in the responsible cellular and molecular mechanisms. In this review, we integrate several robust lines of research: evolutionary trajectories of Xenopus vocalizations, cellular and circuit-level mechanisms of vocalization in selected Xenopus model species, and Xenopus evolutionary history and speciation mechanisms. Integrating recent findings allows us to generate and test specific hypotheses about the evolution of Xenopus vocal circuits. We propose that reduced vocal sex differences in some Xenopus species result from species-specific losses of sexually differentiated neural and neuromuscular features. Modification of sex-hormone-regulated developmental mechanisms is a strong candidate mechanism for reduced vocal sex differences.

Signal recognition particle assembly in relation to the function of amplified nucleoli of Xenopus oocytes.

PubMed

Sommerville, John; Brumwell, Craig L; Politz, Joan C Ritland; Pederson, Thoru

2005-03-15

The signal recognition particle (SRP) is a ribonucleoprotein machine that controls the translation and intracellular sorting of membrane and secreted proteins. The SRP contains a core RNA subunit with which six proteins are assembled. Recent work in both yeast and mammalian cells has identified the nucleolus as a possible initial site of SRP assembly. In the present study, SRP RNA and protein components were identified in the extrachromosomal, amplified nucleoli of Xenopus laevis oocytes. Fluorescent SRP RNA microinjected into the oocyte nucleus became specifically localized in the nucleoli, and endogenous SRP RNA was also detected in oocyte nucleoli by RNA in situ hybridization. An initial step in the assembly of SRP involves the binding of the SRP19 protein to SRP RNA. When green fluorescent protein (GFP)-tagged SRP19 protein was injected into the oocyte cytoplasm it was imported into the nucleus and became concentrated in the amplified nucleoli. After visiting the amplified nucleoli, GFP-tagged SRP19 protein was detected in the cytoplasm in a ribonucleoprotein complex, having a sedimentation coefficient characteristic of the SRP. These results suggest that the amplified nucleoli of Xenopus oocytes produce maternal stores not only of ribosomes, the classical product of nucleoli, but also of SRP, presumably as a global developmental strategy for stockpiling translational machinery for early embryogenesis.

Hyperinnervation improves Xenopus laevis limb regeneration.

PubMed

Mitogawa, Kazumasa; Makanae, Aki; Satoh, Akira

2018-01-15

Xenopus laevis (an anuran amphibian) shows limb regeneration ability between that of urodele amphibians and that of amniotes. Xenopus frogs can initiate limb regeneration but fail to form patterned limbs. Regenerated limbs mainly consist of cone-shaped cartilage without any joints or branches. These pattern defects are thought to be caused by loss of proper expressions of patterning-related genes. This study shows that hyperinnervation surgery resulted in the induction of a branching regenerate. The hyperinnervated blastema allows the identification and functional analysis of the molecules controlling this patterning of limb regeneration. This paper focuses on the nerve affects to improve Xenopus limb patterning ability during regeneration. The nerve molecules, which regulate limb patterning, were also investigated. Blastemas grown in a hyperinnervated forelimb upregulate limb patterning-related genes (shh, lmx1b, and hoxa13). Nerves projecting their axons to limbs express some growth factors (bmp7, fgf2, fgf8, and shh). Inputs of these factors to a blastema upregulated some limb patterning-related genes and resulted in changes in the cartilage patterns in the regenerates. These results indicate that additional nerve factors enhance Xenopus limb patterning-related gene expressions and limb regeneration ability, and that bmp, fgf, and shh are candidate nerve substitute factors. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Histone methyltransferase Dot1L plays a role in postembryonic development in Xenopus tropicalis

PubMed Central

Wen, Luan; Fu, Liezhen; Guo, Xiaogang; Chen, Yonglong; Shi, Yun-Bo

2015-01-01

Histone methylations have been implicated to play important roles in diverse cellular processes. Of particular interest is the methylation of histone H3K79, which is catalyzed by an evolutionarily conserved methyltransferase, disruptor of telomeric silencing (Dot1)-like (Dot1L). To investigate the role of Dot1L during vertebrate development, we have generated a Dot1L-specific transcription activator-like effector nuclease (TALEN) nuclease to knockdown endogenous Dot1L in Xenopus tropicalis, a diploid species highly related to the well-known developmental model Xenopus laevis, a pseudotetraploid amphibian. We show that the TALEN was extremely efficient in mutating Dot1L when expressed in fertilized eggs, creating essentially Dot1L knockout embryos with little H3K79 methylation. Importantly, we observed that Dot1L knockdown had no apparent effect on embryogenesis because normally feeding tadpoles were formed, consistent with the lack of maternal Dot1L expression. On the other hand, Dot1L knockdown severely retarded the growth of the tadpoles and led to tadpole lethality prior to metamorphosis. These findings suggest that Dot1L and H3K79 methylation play an important role for tadpole growth and development prior to metamorphosis into a frog. Our findings further reveal interesting similarities and differences between Xenopus and mouse development and suggest the existence of 2 separate phases of vertebrate development with distinct requirements for epigenetic modifications.—Wen, L., Fu, L., Guo, X., Chen, Y., Shi, Y.-B. Histone methyltransferase Dot1L plays a role in postembryonic development in Xenopus tropicalis. PMID:25366346

Crystallization and X-ray diffraction analysis of the CH domain of the cotton kinesin GhKCH2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin, Xinghua; The Fourth Military Medical University, No. 169 Changlexi Road, Xincheng District, Xi’an 710032, People’s Republic of; Chen, Ziwei

The cloning, expression, purification and crystallization of the CH domain of the plant-specific kinesin GhKCH2 is reported. GhKCH2 belongs to a group of plant-specific kinesins (KCHs) containing an actin-binding calponin homology (CH) domain in the N-terminus. Previous studies revealed that the GhKCH2 CH domain (GhKCH2-CH) had a higher affinity for F-actin (K{sub d} = 0.42 ± 0.02 µM) than most other CH-domain-containing proteins. To understand the underlying mechanism, prokaryotically expressed GhKCH2-CH (amino acids 30–166) was purified and crystallized. Crystals were grown by the sitting-drop vapour-diffusion method using 0.1 M Tris–HCl pH 7.0, 20%(w/v) PEG 8000 as a precipitant. The crystalsmore » diffracted to a resolution of 2.5 Å and belonged to space group P2{sub 1}, with unit-cell parameters a = 41.57, b = 81.92, c = 83.00 Å, α = 90.00, β = 97.31, γ = 90.00°. Four molecules were found in the asymmetric unit with a Matthews coefficient of 2.22 Å{sup 3} Da{sup −1}, corresponding to a solvent content of 44.8%.« less

Xenopus egg cytoplasm with intact actin.

PubMed

Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

2014-01-01

We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. © 2014 Elsevier Inc. All rights reserved.

Effects of S-adenosylmethionine decarboxylase, polyamines, amino acids, and weak bases (amines and ammonia) on development and ribosomal RNA synthesis in Xenopus embryos.

PubMed

Shiokawa, Koichiro; Aso, Mai; Kondo, Takeshi; Takai, Jun-Ichi; Yoshida, Junki; Mishina, Takamichi; Fuchimukai, Kota; Ogasawara, Tsukasa; Kariya, Taro; Tashiro, Kosuke; Igarashi, Kazuei

2010-02-01

We have been studying control mechanisms of gene expression in early embryogenesis in a South African clawed toad Xenopus laevis, especially during the period of midblastula transition (MBT), or the transition from the phase of active cell division (cleavage stage) to the phase of extensive morphogenesis (post-blastular stages). We first found that ribosomal RNA synthesis is initiated shortly after MBT in Xenopus embryos and those weak bases, such as amines and ammonium ion, selectively inhibit the initiation and subsequent activation of rRNA synthesis. We then found that rapidly labeled heterogeneous mRNA-like RNA is synthesized in embryos at pre-MBT stage. We then performed cloning and expression studies of several genes, such as those for activin receptors, follistatin and aldolases, and then reached the studies of S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine metabolism. Here, we cloned a Xenopus SAMDC cDNA and performed experiments to overexpress the in vitro-synthesized SAMDC mRNA in Xenopus early embryos, and found that the maternally preset program of apoptosis occurs in cleavage stage embryos, which is executed when embryos reach the stage of MBT. In the present article, we first summarize results on SAMDC and the maternal program of apoptosis, and then describe our studies on small-molecular-weight substances like polyamines, amino acids, and amines in Xenopus embryos. Finally, we summarize our studies on weak bases, especially on ammonium ion, as the specific inhibitor of ribosomal RNA synthesis in Xenopus embryonic cells.

Xenopus origin recognition complex (ORC) initiates DNA replication preferentially at sequences targeted by Schizosaccharomyces pombe ORC

PubMed Central

Kong, Daochun; Coleman, Thomas R.; DePamphilis, Melvin L.

2003-01-01

Budding yeast (Saccharomyces cerevisiae) origin recognition complex (ORC) requires ATP to bind specific DNA sequences, whereas fission yeast (Schizosaccharomyces pombe) ORC binds to specific, asymmetric A:T-rich sites within replication origins, independently of ATP, and frog (Xenopus laevis) ORC seems to bind DNA non-specifically. Here we show that despite these differences, ORCs are functionally conserved. Firstly, SpOrc1, SpOrc4 and SpOrc5, like those from other eukaryotes, bound ATP and exhibited ATPase activity, suggesting that ATP is required for pre-replication complex (pre-RC) assembly rather than origin specificity. Secondly, SpOrc4, which is solely responsible for binding SpORC to DNA, inhibited up to 70% of XlORC-dependent DNA replication in Xenopus egg extract by preventing XlORC from binding to chromatin and assembling pre-RCs. Chromatin-bound SpOrc4 was located at AT-rich sequences. XlORC in egg extract bound preferentially to asymmetric A:T-sequences in either bare DNA or in sperm chromatin, and it recruited XlCdc6 and XlMcm proteins to these sequences. These results reveal that XlORC initiates DNA replication preferentially at the same or similar sites to those targeted in S.pombe. PMID:12840006

Integrated regulation of motor-driven organelle transport by scaffolding proteins.

PubMed

Fu, Meng-meng; Holzbaur, Erika L F

2014-10-01

Intracellular trafficking pathways, including endocytosis, autophagy, and secretion, rely on directed organelle transport driven by the opposing microtubule motor proteins kinesin and dynein. Precise spatial and temporal targeting of vesicles and organelles requires the integrated regulation of these opposing motors, which are often bound simultaneously to the same cargo. Recent progress demonstrates that organelle-associated scaffolding proteins, including Milton/TRAKs (trafficking kinesin-binding protein), JIP1, JIP3 (JNK-interacting proteins), huntingtin, and Hook1, interact with molecular motors to coordinate activity and sustain unidirectional transport. Scaffolding proteins also bind to upstream regulatory proteins, including kinases and GTPases, to modulate transport in the cell. This integration of regulatory control with motor activity allows for cargo-specific changes in the transport or targeting of organelles in response to cues from the complex cellular environment. Copyright © 2014 Elsevier Ltd. All rights reserved.

Slowed Relaxation in Fatigued Skeletal Muscle Fibers of Xenopus and Mouse

PubMed Central

Westerblad, Håkan; Lännergren, Jan; Allen, David G.

1997-01-01

Slowing of relaxation is an important characteristic of skeletal muscle fatigue. The aim of the present study was to quantify the relative contribution of altered Ca2+ handling (calcium component) and factors down-stream to Ca2+ (cross-bridge component) to the slowing of relaxation in fatigued fibers of Xenopus and mouse. Two types of Xenopus fibers were used: easily fatigued, type 1 fibers and fatigue resistant, type 2 fibers. In these Xenopus fibers the free myoplasmic [Ca2+] ([Ca2+]i) was measured with indo-1, and the relaxation of Ca2+-derived force, constructed from tetanic [Ca2+]i records and in vivo [Ca2+]i-force curves, was analyzed. An alternative method was used in both Xenopus and mouse fibers: fibers were rapidly shortened during the initial phase of relaxation, and the time to the peak of force redevelopment was measured. These two methods gave similar results and showed proportional slowing of the calcium and cross-bridge components of relaxation in both fatigued type 1 and type 2 Xenopus fibers, whereas only the cross-bridge component was slowed in fatigued mouse fibers. Ca2+ removal from the myoplasm during relaxation was markedly less effective in Xenopus fibers as compared to mouse fibers. Fatigued Xenopus fibers displayed a reduced rate of sarcoplasmic reticulum Ca2+ uptake and increased sarcoplasmic reticulum Ca2+ leak. Some fibers were stretched at various times during relaxation. The resistance to these stretches was increased during fatigue, especially in Xenopus fibers, which indicates that longitudinal movements during relaxation had become less pronounced and this might contribute to the increased cross-bridge component of relaxation in fatigue. In conclusion, slowing of relaxation in fatigued Xenopus fibers is caused by impaired Ca2+ handling and altered cross-bridge kinetics, whereas the slowing in mouse fibers is only due to altered cross-bridge kinetics. PMID:9089444

Effect of diadenosine tetraphosphate microinjection on heat shock protein synthesis in Xenopus laevis oocytes.

PubMed Central

Guedon, G; Sovia, D; Ebel, J P; Befort, N; Remy, P

1985-01-01

Bisnucleosides polyphosphates are thought to be chemical messengers signalling to the cell the onset of various stresses. Diadenosine tri- and tetraphosphates (respectively, Ap3A and Ap4A) accumulate in prokaryotic and eukaryotic cells under heat shock conditions, suggesting they could trigger the synthesis of heat shock proteins (hsps). In this study, Ap4A, Ap3A and, as a control, Ap4 (adenosine tetraphosphate) were injected into Xenopus oocytes. Whereas none of these compounds is able to trigger the synthesis of hsps in the absence of hyperthermic treatment, nuclear microinjection of Ap4A after a mild heat shock specifically enhances the synthesis of the 70-kd hsp, which is involved in the regulation and possibly the termination of the heat shock response. The microinjection of Ap4A prior to the hyperthermic treatment results in a strong inhibition of hsps synthesis (with the exception of the 70-kd hsp) suggesting that Ap4A is involved in the regulation and/or termination of the heat shock response. Ap3A and Ap4 do not induce any detectable modification of hsps expression. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. PMID:4092696

Transcription factor COUP-TFII is indispensable for venous and lymphatic development in zebrafish and Xenopus laevis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aranguren, Xabier L., E-mail: xabier.lopezaranguren@med.kuleuven.be; Beerens, Manu, E-mail: manu.beerens@med.kuleuven.be; Vandevelde, Wouter, E-mail: woutervandevelde@gmail.com

Highlights: {yields} COUP-TFII deficiency in zebrafish affects arterio-venous EC specification. {yields} COUP-TFII is indispensable for lymphatic development in zebrafish. {yields} COUP-TFII knockdown in Xenopus disrupts lymphatic EC differentiation and migration. {yields} COUP-TFII's role in EC fate decisions is evolutionary conserved. -- Abstract: Transcription factors play a central role in cell fate determination. Gene targeting in mice revealed that Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII, also known as Nuclear Receptor 2F2 or NR2F2) induces a venous phenotype in endothelial cells (ECs). More recently, NR2F2 was shown to be required for initiating the expression of Prox1, responsible for lymphatic commitment ofmore » venous ECs. Small animal models like zebrafish embryos and Xenopus laevis tadpoles have been very useful to elucidate mechanisms of (lymph) vascular development. Therefore, the role of NR2F2 in (lymph) vascular development was studied by eliminating its expression in these models. Like in mice, absence of NR2F2 in zebrafish resulted in distinct vascular defects including loss of venous marker expression, major trunk vessel fusion and vascular leakage. Both in zebrafish and Xenopus the development of the main lymphatic structures was severely hampered. NR2F2 knockdown significantly decreased prox1 expression in zebrafish ECs and the same manipulation affected lymphatic (L)EC commitment, migration and function in Xenopus tadpoles. Therefore, the role of NR2F2 in EC fate determination is evolutionary conserved.« less

Ciliary targeting of olfactory CNG channels requires the CNGB1b subunit and the kinesin-2 motor protein, KIF17.

PubMed

Jenkins, Paul M; Hurd, Toby W; Zhang, Lian; McEwen, Dyke P; Brown, R Lane; Margolis, Ben; Verhey, Kristen J; Martens, Jeffrey R

2006-06-20

Nonmotile cilia on olfactory sensory neurons (OSNs) compartmentalize signaling molecules, including odorant receptors and cyclic nucleotide-gated (CNG) channels, allowing for efficient, spatially confined responses to sensory stimuli . Little is known about the mechanisms of the ciliary targeting of olfactory CNG channels, composed of three subunits: CNGA2, CNGA4, and CNGB1b . Recent reports suggest that subunit composition of the retinal CNG channel influences localization, leading to disease . However, the mechanistic role of subunits in properly targeting native olfactory CNG channels remains unclear. Here, we show that heteromeric assembly with CNGB1b, containing a critical carboxy-terminal motif (RVxP), is required for ciliary trafficking of olfactory CNG channels. Movement of proteins within the cilia is governed by intraflagellar transport (IFT), a process that facilitates bidirectional movement of cargo along microtubules. Work in C. elegans has established that heterotrimeric and homodimeric kinesin-2 family members play a critical role in anterograde transport . In mammalian systems, the heterotrimeric KIF3a/KIF3b/KAP-3 complex plays a clear role in IFT; however, no role has been established for KIF17, the mammalian homolog of OSM-3 . Here, we demonstrate that KIF17 is required for olfactory CNG channel targeting, providing novel insights into mechanisms of mammalian ciliary transport.

The RNA-binding protein xCIRP2 is involved in apoptotic tail regression during metamorphosis in Xenopus laevis tadpoles.

PubMed

Eto, Ko; Iwama, Tomoyuki; Tajima, Tatsuya; Abe, Shin-ichi

2012-10-01

Frog metamorphosis induced by thyroid hormone (TH) involves not only cell proliferation and differentiation in reconstituted organs such as limbs, but also apoptotic cell death in degenerated organs such as tails. However, the molecular mechanisms directing the TH-dependent cell fate determination remain unclear. We have previously identified from newts an RNA-binding protein (nRBP) acting as the regulator governing survival and death in germ cells during spermatogenesis. To investigate the molecular events leading the tail resorption during metamorphosis, we analyzed the expression, the functional role in apoptosis, and the regulation of xCIRP2, a frog homolog of nRBP, in tails of Xenopus laevis tadpoles. At the prometamorphic stage, xCIRP2 protein is expressed in fibroblast, epidermal, nerve, and muscular cells and localized in their cytoplasm. When spontaneous metamorphosis progressed, the level of xCIRP2 mRNA remained unchanged but the amount of the protein decreased. In organ cultures of tails at the prometamorphic stage, xCIRP2 protein decreased before their lengths shortened during TH-dependent metamorphosis. The inhibition of calpain or proteasome attenuated the TH-induced decrease of xCIRP2 protein in tails, impairing their regression. These results suggest that xCIRP2 protein is downregulated through calpain- and proteasome-mediated proteolysis in response to TH at the onset of metamorphosis, inducing apoptosis in tails and thereby degenerating them. Copyright © 2012 Elsevier Inc. All rights reserved.

What we can learn from a tadpole about ciliopathies and airway diseases: Using systems biology in Xenopus to study cilia and mucociliary epithelia.

PubMed

Walentek, Peter; Quigley, Ian K

2017-01-01

Over the past years, the Xenopus embryo has emerged as an incredibly useful model organism for studying the formation and function of cilia and ciliated epithelia in vivo. This has led to a variety of findings elucidating the molecular mechanisms of ciliated cell specification, basal body biogenesis, cilia assembly, and ciliary motility. These findings also revealed the deep functional conservation of signaling, transcriptional, post-transcriptional, and protein networks employed in the formation and function of vertebrate ciliated cells. Therefore, Xenopus research can contribute crucial insights not only into developmental and cell biology, but also into the molecular mechanisms underlying cilia related diseases (ciliopathies) as well as diseases affecting the ciliated epithelium of the respiratory tract in humans (e.g., chronic lung diseases). Additionally, systems biology approaches including transcriptomics, genomics, and proteomics have been rapidly adapted for use in Xenopus, and broaden the applications for current and future translational biomedical research. This review aims to present the advantages of using Xenopus for cilia research, highlight some of the evolutionarily conserved key concepts and mechanisms of ciliated cell biology that were elucidated using the Xenopus model, and describe the potential for Xenopus research to address unresolved questions regarding the molecular mechanisms of ciliopathies and airway diseases. © 2017 Wiley Periodicals, Inc.

Anosmin-1 is essential for neural crest and cranial placodes formation in Xenopus.

PubMed

Bae, Chang-Joon; Hong, Chang-Soo; Saint-Jeannet, Jean-Pierre

2018-01-15

During embryogenesis vertebrates develop a complex craniofacial skeleton associated with sensory organs. These structures are primarily derived from two embryonic cell populations the neural crest and cranial placodes, respectively. Neural crest cells and cranial placodes are specified through the integrated action of several families of signaling molecules, and the subsequent activation of a complex network of transcription factors. Here we describe the expression and function of Anosmin-1 (Anos1), an extracellular matrix protein, during neural crest and cranial placodes development in Xenopus laevis. Anos1 was identified as a target of Pax3 and Zic1, two transcription factors necessary and sufficient to generate neural crest and cranial placodes. Anos1 is expressed in cranial neural crest progenitors at early neurula stage and in cranial placode derivatives later in development. We show that Anos1 function is required for neural crest and sensory organs development in Xenopus, consistent with the defects observed in Kallmann syndrome patients carrying a mutation in ANOS1. These findings indicate that anos1 has a conserved function in the development of craniofacial structures, and indicate that anos1-depleted Xenopus embryos represent a useful model to analyze the pathogenesis of Kallmann syndrome. Copyright © 2017. Published by Elsevier Inc.

Processivity of the Kinesin-2 KIF3A Results from Rear Head Gating and Not Front Head Gating*

PubMed Central

Chen, Geng-Yuan; Arginteanu, David F. J.; Hancock, William O.

2015-01-01

The kinesin-2 family motor KIF3A/B works together with dynein to bidirectionally transport intraflagellar particles, melanosomes, and neuronal vesicles. Compared with kinesin-1, kinesin-2 is less processive, and its processivity is more sensitive to load, suggesting that processivity may be controlled by different gating mechanisms. We used stopped-flow and steady-state kinetics experiments, along with single-molecule and multimotor assays to characterize the entire kinetic cycle of a KIF3A homodimer that exhibits motility similar to that of full-length KIF3A/B. Upon first encounter with a microtubule, the motor rapidly exchanges both mADP and mATP. When adenosine 5′-[(β,γ)-imido]triphosphate was used to entrap the motor in a two-head-bound state, exchange kinetics were unchanged, indicating that rearward strain in the two-head-bound state does not alter nucleotide binding to the front head. A similar lack of front head gating was found when intramolecular strain was enhanced by shortening the neck linker domain from 17 to 14 residues. In single-molecule assays in ADP, the motor dissociates at 2.1 s−1, 20-fold slower than the stepping rate, demonstrating the presence of rear head gating. In microtubule pelleting assays, the KDMt is similar in ADP and ATP. The data and accompanying simulations suggest that, rather than KIF3A processivity resulting from strain-dependent regulation of nucleotide binding (front head gating), the motor spends a significant fraction of its hydrolysis cycle in a low affinity state but dissociates only slowly from this state. This work provides a mechanism to explain differences in the load-dependent properties of kinesin-1 and kinesin-2. PMID:25657001

Seeing the future: using Xenopus to understand eye regeneration.

PubMed

Tseng, Ai-Sun

2017-01-01

Studies of Xenopus eye development have contributed considerably to the understanding of vertebrate neurogenesis, including eye field specification, cell fate determination and identification of genes critical for eye formation. This knowledge has served as a solid foundation for cellular and molecular examinations of the robust regenerative capacity of the Xenopus eye. The retina, lens, and the optic nerve are capable of regeneration after injury in both larval and adult stages. Here, we discuss the current models for studying eye regeneration in Xenopus and their potential applications for providing insights into human eye diseases. As Xenopus has many of the same tools that are available for other regeneration models, we thus highlight the distinct strengths and versatility of this organism that make it especially suited for extrapolating and testing strategies aimed at promoting regeneration and repair in eye tissues. Furthermore, we outline a promising future for the use of new techniques and approaches to address outstanding questions in understanding eye regeneration. © 2017 Wiley Periodicals, Inc.

RNA localization in Xenopus oocytes uses a core group of trans-acting factors irrespective of destination.

PubMed

Snedden, Donald D; Bertke, Michelle M; Vernon, Dominic; Huber, Paul W

2013-07-01

The 3' untranslated region of mRNA encoding PHAX, a phosphoprotein required for nuclear export of U-type snRNAs, contains cis-acting sequence motifs E2 and VM1 that are required for localization of RNAs to the vegetal hemisphere of Xenopus oocytes. However, we have found that PHAX mRNA is transported to the opposite, animal, hemisphere. A set of proteins that cross-link to the localization elements of vegetally localized RNAs are also cross-linked to PHAX and An1 mRNAs, demonstrating that the composition of RNP complexes that form on these localization elements is highly conserved irrespective of the final destination of the RNA. The ability of RNAs to bind this core group of proteins is correlated with localization activity. Staufen1, which binds to Vg1 and VegT mRNAs, is not associated with RNAs localized to the animal hemisphere and may determine, at least in part, the direction of RNA movement in Xenopus oocytes.

Characterizing and modeling protein-surface interactions in lab-on-chip devices

NASA Astrophysics Data System (ADS)

Katira, Parag

Protein adsorption on surfaces determines the response of other biological species present in the surrounding solution. This phenomenon plays a major role in the design of biomedical and biotechnological devices. While specific protein adsorption is essential for device function, non-specific protein adsorption leads to the loss of device function. For example, non-specific protein adsorption on bioimplants triggers foreign body response, in biosensors it leads to reduced signal to noise ratios, and in hybrid bionanodevices it results in the loss of confinement and directionality of molecular shuttles. Novel surface coatings are being developed to reduce or completely prevent the non-specific adsorption of proteins to surfaces. A novel quantification technique for extremely low protein coverage on surfaces has been developed. This technique utilizes measurement of the landing rate of microtubule filaments on kinesin proteins adsorbed on a surface to determine the kinesin density. Ultra-low limits of detection, dynamic range, ease of detection and availability of a ready-made kinesin-microtubule kit makes this technique highly suitable for detecting protein adsorption below the detection limits of standard techniques. Secondly, a random sequential adsorption model is presented for protein adsorption to PEO-coated surfaces. The derived analytical expressions accurately predict the observed experimental results from various research groups, suggesting that PEO chains act as almost perfect steric barriers to protein adsorption. These expressions can be used to predict the performance of a variety of systems towards resisting protein adsorption and can help in the design of better non-fouling surface coatings. Finally, in biosensing systems, target analytes are captured and concentrated on specifically adsorbed proteins for detection. Non-specific adsorption of proteins results in the loss of signal, and an increase in the background. The use of nanoscale transducers as

Molecular characterization of a KIF3B-like kinesin gene in the testis of Octopus tankahkeei (Cephalopoda, Octopus).

PubMed

Dang, Ran; Zhu, Jun-Quan; Tan, Fu-Qing; Wang, Wei; Zhou, Hong; Yang, Wan-Xi

2012-05-01

KIF3B is known for maintaining and assembling cilia and flagellum. To date, the function of KIF3B and its relationship with KIF3A during spermiogenesis in the cephalopod Octopus tankahkeei remains unknown. In the present study, we characterized a gene encoding a homologue of rat KIF3B in the O. tankahkeei testis and examined its temporal and spatial expression pattern during spermiogenesis. The cDNA of KIF3B was obtained with degenerate and RACE PCR and the distribution pattern of ot-kif3b were observed with RT-PCR. The morphological development during spermiogenesis was illustrated by histological and transmission electron microscopy and mRNA expression of ot-kif3b was observed by in situ hybridization. The 2,365 nucleotides cDNA consisted of a 102 bp 5' untranslated region (UTR), a 2,208 bp open reading frame (ORF) encoding a protein of 736 amino acids, and a 55 bp 3' UTR. Multiple alignments revealed that the putative Ot-KIF3B shared 68, 68, 69, 68, and 67% identity with that of Homo sapiens, Mus musculus, Gallus gallus, Danio rerio, and Xenopus laevis, respectively, along with high identities with Ot-KIF3A in fundamental structures. Ot-kif3b transcripts appeared gradually in early spermatids, increased in intermediate spermatids and maximized in drastically remodeled and final spermatids. The kif3b gene is identified and its expression pattern is demonstrated for the first time in O. tankahkeei. Compared to ot-kif3a reported by our laboratory before, our data suggested that the putative heterodimeric motor proteins Ot-KIF3A/B may be involved in intraspermatic transport and might contribute to structural changes during spermiogenesis.

Identification of a candidate CD5 homologue in the amphibian Xenopus laevis.

PubMed

Jürgens, J B; Gartland, L A; Du Pasquier, L; Horton, J D; Göbel, T W; Cooper, M D

1995-11-01

We identified a novel T cell Ag in the South African clawed toad (Xenopus laevis) by a mAb designated 2B1. This Ag is present in relatively high levels on most thymocytes, approximately 65% of splenocytes, 55% of PBL, and 65% of intestinal lymphocytes, but is rarely seen on IgM+ B cells in any of these tissues. Lymphocytes bearing the 2B1 Ag proliferate in response to stimulation with Con A or PHA, whereas the 2B1- lymphocytes are reactive to LPS. Biochemical analysis indicates that this Ag is a differentially phosphorylated glycoprotein of 71 to 82 kDa. The protein core of 64 kDa bears both N- and O-linked carbohydrate side chains. The amino-terminal protein sequence of the 2B1 Ag shares significant homology with both the macrophage scavenger receptor type 1 motif and the mammalian CD5/CD6 family. The biochemical characteristics and cellular distribution of the 2B1 Ag suggest that it represents the CD5 homologue in X. laevis. While T cells constitutively express this highly conserved molecule, Xenopus B cells acquire the CD5 homologue only when they are stimulated in the presence of T cells.

Controlling the Messenger: Regulated Translation of Maternal mRNAs in Xenopus laevis Development

PubMed Central

Fox, Catherine A.; Dowdle, Megan E.; Blaser, Susanne Imboden; Chung, Andy; Park, Sookhee

2017-01-01

The selective translation of maternal mRNAs encoding cell-fate determinants drives the earliest decisions of embryogenesis that establish the vertebrate body plan. This chapter will discuss studies in Xenopus laevis that provide insights into mechanisms underlying this translational control. Xenopus has been a powerful model organism for many discoveries relevant to the translational control of maternal mRNAs because of the large size of its oocytes and eggs that allow for microinjection of molecules and the relative ease of manipulating the oocyte to egg transition (maturation) and fertilization in culture. Consequently, many key studies have focused on the expression of maternal mRNAs during the oocyte to egg transition (the meiotic cell cycle) and the rapid cell divisions immediately following fertilization. This research has made seminal contributions to our understanding of translational regulatory mechanisms, but while some of the mRNAs under consideration at these stages encode cell-fate determinants, many encode cell cycle regulatory proteins that drive these early cell cycles. In contrast, while maternal mRNAs encoding key developmental (i.e., cell-fate) regulators that function after the first cleavage stages may exploit aspects of these foundational mechanisms, studies reveal that these mRNAs must also rely on distinct and, as of yet, incompletely understood mechanisms. These findings are logical because the functions of such developmental regulatory proteins have requirements distinct from cell cycle regulators, including becoming relevant only after fertilization and then only in specific cells of the embryo. Indeed, key maternal cell-fate determinants must be made available in exquisitely precise amounts (usually low), only at specific times and in specific cells during embryogenesis. To provide an appreciation for the regulation of maternal cell-fate determinant expression, an overview of the maternal phase of Xenopus embryogenesis will be presented

Fission yeast cells overproducing HSET/KIFC1 provides a useful tool for identification and evaluation of human kinesin-14 inhibitors.

PubMed

Yukawa, Masashi; Yamauchi, Tomoaki; Kurisawa, Naoaki; Ahmed, Shakil; Kimura, Ken-Ichi; Toda, Takashi

2018-07-01

Many human cancer cells contain more than two centrosomes, yet these cancer cells can form pseudo-bipolar spindles through the mechanism, called centrosome clustering, and survive, instead of committing lethal multipolar mitoses. Kinesin-14/HSET, a minus end-directed motor, plays a crucial role in centrosome clustering. Accordingly, HSET is deemed to be a promising chemotherapeutic target to selectively kill cancer cells. Recently, three HSET inhibitors (AZ82, CW069 and SR31527) have been reported, but their specificity and efficacy have not been evaluated rigorously. This downside partly stems from the lack of robust systems for the assessment of these drugs. Yeasts and filamentous fungi provide not only powerful models for basic and applied biology but also versatile tools for drug discovery and evaluation. Here we show that these three inhibitors on their own are cytotoxic to fission yeast, suggesting that they have off-targets in vivo except for kinesin-14. Nonetheless, intriguingly, AZ82 can neutralize otherwise toxic overproduced HSET; this includes a substantial reduction in the percentage of HSET-driven abnormal mitotic cells and partial suppression of its lethality. SR31527 also displays modest neutralizing activity, while we do not detect such activity in CW069. As an experimental proof-of-principle study, we have treated HSET-overproducing fission yeast cells with extracts prepared from various plant species and found activities that rescue HSET-driven lethality in those from Chamaecyparis pisifera and Toxicodendron trichocarpum. This methodology of protein overproduction in fission yeast, therefore, provides a convenient, functional assay system by which to screen for not only selective human kinesin-14 inhibitors but also those against other molecules of interest. Copyright © 2018 Elsevier Inc. All rights reserved.

How does the Xenopus laevis embryonic cell cycle avoid spatial chaos?

PubMed Central

Gelens, Lendert; Huang, Kerwyn Casey; Ferrell, James E.

2015-01-01

Summary Theoretical studies have shown that a deterministic biochemical oscillator can become chaotic when operating over a sufficiently large volume, and have suggested that the Xenopus laevis cell cycle oscillator operates close to such a chaotic regime. To experimentally test this hypothesis, we decreased the speed of the post-fertilization calcium wave, which had been predicted to generate chaos. However, cell divisions were found to develop normally and eggs developed into normal tadpoles. Motivated by these experiments, we carried out modeling studies to understand the prerequisites for the predicted spatial chaos. We showed that this type of spatial chaos requires oscillatory reaction dynamics with short pulse duration, and postulated that the mitotic exit in Xenopus laevis is likely slow enough to avoid chaos. In systems with shorter pulses, chaos may be an important hazard, as in cardiac arrhythmias, or a useful feature, as in the pigmentation of certain mollusk shells. PMID:26212326

Single Molecule and Collective Dynamics of Motor Protein Coupled with Mechano-Sensitive Chemical Reaction

NASA Astrophysics Data System (ADS)

Iwaki, Mitsuhiro; Marcucci, Lorenzo; Togashi, Yuichi; Yanagida, Toshio

2013-12-01

Motor proteins such as myosin and kinesin hydrolyze ATP into ADP and Pi to convert chemical energy into mechanical work. This resultsin various motile processes like muscle contraction, vesicle transport and cell division. Recent single molecule experiments have revealed that external load applied to these motor proteins perturb not only the mechanical motion, but the ATP hydrolysis cycle as well, making these molecules mechano-enzymes. Here, we describe our single molecule detection techniques to reveal the mechano-enzymatic properties of myosin and introduce recent progress from both experimental and theoretical approaches at the single- and multiple-molecule level.

Physical limits on kinesin-5–mediated chromosome congression in the smallest mitotic spindles

PubMed Central

McCoy, Kelsey M.; Tubman, Emily S.; Claas, Allison; Tank, Damien; Clancy, Shelly Applen; O’Toole, Eileen T.; Berman, Judith; Odde, David J.

2015-01-01

A characteristic feature of mitotic spindles is the congression of chromosomes near the spindle equator, a process mediated by dynamic kinetochore microtubules. A major challenge is to understand how precise, submicrometer-scale control of kinetochore micro­tubule dynamics is achieved in the smallest mitotic spindles, where the noisiness of microtubule assembly/disassembly will potentially act to overwhelm the spatial information that controls microtubule plus end–tip positioning to mediate congression. To better understand this fundamental limit, we conducted an integrated live fluorescence, electron microscopy, and modeling analysis of the polymorphic fungal pathogen Candida albicans, which contains one of the smallest known mitotic spindles (<1 μm). Previously, ScCin8p (kinesin-5 in Saccharomyces cerevisiae) was shown to mediate chromosome congression by promoting catastrophe of long kinetochore microtubules (kMTs). Using C. albicans yeast and hyphal kinesin-5 (Kip1p) heterozygotes (KIP1/kip1∆), we found that mutant spindles have longer kMTs than wild-type spindles, consistent with a less-organized spindle. By contrast, kinesin-8 heterozygous mutant (KIP3/kip3∆) spindles exhibited the same spindle organization as wild type. Of interest, spindle organization in the yeast and hyphal states was indistinguishable, even though yeast and hyphal cell lengths differ by two- to fivefold, demonstrating that spindle length regulation and chromosome congression are intrinsic to the spindle and largely independent of cell size. Together these results are consistent with a kinesin-5–mediated, length-dependent depolymerase activity that organizes chromosomes at the spindle equator in C. albicans to overcome fundamental noisiness in microtubule self-assembly. More generally, we define a dimensionless number that sets a fundamental physical limit for maintaining congression in small spindles in the face of assembly noise and find that C. albicans operates very close to

Prokaryotic Ubiquitin-Like Protein Modification

PubMed Central

Maupin-Furlow, Julie A.

2016-01-01

Prokaryotes form ubiquitin (Ub)-like isopeptide bonds on the lysine residues of proteins by at least two distinct pathways that are reversible and regulated. In mycobacteria, the C-terminal Gln of Pup (prokaryotic ubiquitin-like protein) is deamidated and isopeptide linked to proteins by a mechanism distinct from ubiquitylation in enzymology yet analogous to ubiquitylation in targeting proteins for destruction by proteasomes. Ub-fold proteins of archaea (SAMPs, small archaeal modifier proteins) and Thermus (TtuB, tRNA-two-thiouridine B) that differ from Ub in amino acid sequence, yet share a common β-grasp fold, also form isopeptide bonds by a mechanism that appears streamlined compared with ubiquitylation. SAMPs and TtuB are found to be members of a small group of Ub-fold proteins that function not only in protein modification but also in sulfur-transfer pathways associated with tRNA thiolation and molybdopterin biosynthesis. These multifunctional Ub-fold proteins are thought to be some of the most ancient of Ub-like protein modifiers. PMID:24995873

Discovery of (+)-N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide (AZD4877), a kinesin spindle protein inhibitor and potential anticancer agent.

PubMed

Theoclitou, Maria-Elena; Aquila, Brian; Block, Michael H; Brassil, Patrick J; Castriotta, Lillian; Code, Erin; Collins, Michael P; Davies, Audrey M; Deegan, Tracy; Ezhuthachan, Jayachandran; Filla, Sandra; Freed, Ellen; Hu, Haiqing; Huszar, Dennis; Jayaraman, Muthusamy; Lawson, Deborah; Lewis, Paula M; Nadella, Murali V P; Oza, Vibha; Padmanilayam, Maniyan; Pontz, Timothy; Ronco, Lucienne; Russell, Daniel; Whitston, David; Zheng, Xiaolan

2011-10-13

Structure-activity relationship analysis identified (+)-N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide (AZD4877), from a series of novel kinesin spindle protein (KSP) inhibitors, as exhibiting both excellent biochemical potency and pharmaceutical properties suitable for clinical development. The selected compound arrested cells in mitosis leading to the formation of the monopolar spindle phenotype characteristic of KSP inhibition and induction of cellular death. A favorable pharmacokinetic profile and notable in vivo efficacy supported the selection of this compound as a clinical candidate for the treatment of cancer.

Characterization of X-OCRL, a Xenopus laevis homologue of OCRL-1, the Lowe oculocerebrorenal syndrome candidate gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reilly, D.S.; Nussbaum, R.L.

1994-09-01

The Lowe oculocerebrorenal syndrome (OCRL) is an X-linked disease characterized by congenital cataract, mental retardation, and renal tubular dysfunction. A candidate cDNA, OCRL-1, was identified by positional cloning and mutations in OCRL-1 have been detected in patients with Lowe syndrome. The OCRL-1 nucleotide sequence encodes a predicted protein of 968 amino acids and shares 51% amino acid identity with a human inositol polyphosphate-5-phosphatase. This suggests that the underlying defect in OCRL may be due to a defect in inositol phosphate metabolism. The isolation of OCRL-1 provides the opportunity to investigate its function through the use of animal model systems. Wemore » have isolated a partial cDNA clone encoding an OCRL-1 homologue, X-OCRL, from the South African clawed frog, Xenopus laevis. We used a portion of the human cDNA to screen a Xenopus laevis embryo cDNA library and isolated four positive clones. One clone, 42-5A, is a 650 bp insert with over 75% amino acid identity to the corresponding region of the human OCRL-1 sequence. 42-5A detects messenger RNA in adult Xenopus brain, stomach, small intestine, skin, muscle, lung, blood, and oviduct. X-OCRL messenger RNA is first detected during late gastrula and continues to be expressed throughout Xenopus development. In situ hybridization studies are underway to identify the cellular localization of X-OCRL expression in Xenopus embryos and adult tissues. We are especially interested in characterizing X-OCRL expression during formation of the amphibian lens since congenital cataracts are a constant feature of the human disease.« less

CENP-E Kinesin Interacts with SKAP Protein to Orchestrate Accurate Chromosome Segregation in Mitosis*

PubMed Central

Huang, Yuejia; Wang, Wenwen; Yao, Phil; Wang, Xiwei; Liu, Xing; Zhuang, Xiaoxuan; Yan, Feng; Zhou, Jinhua; Du, Jian; Ward, Tarsha; Zou, Hanfa; Zhang, Jiancun; Fang, Guowei; Ding, Xia; Dou, Zhen; Yao, Xuebiao

2012-01-01

Mitotic chromosome segregation is orchestrated by the dynamic interaction of spindle microtubules with the kinetochore. Although previous studies show that the mitotic kinesin CENP-E forms a link between attachment of the spindle microtubule to the kinetochore and the mitotic checkpoint signaling cascade, the molecular mechanism underlying dynamic kinetochore-microtubule interactions in mammalian cells remains elusive. Here, we identify a novel interaction between CENP-E and SKAP that functions synergistically in governing dynamic kinetochore-microtubule interactions. SKAP binds to the C-terminal tail of CENP-E in vitro and is essential for an accurate kinetochore-microtubule attachment in vivo. Immunoelectron microscopic analysis indicates that SKAP is a constituent of the kinetochore corona fibers of mammalian centromeres. Depletion of SKAP or CENP-E by RNA interference results in a dramatic reduction of inter-kinetochore tension, which causes chromosome mis-segregation with a prolonged delay in achieving metaphase alignment. Importantly, SKAP binds to microtubules in vitro, and this interaction is synergized by CENP-E. Based on these findings, we propose that SKAP cooperates with CENP-E to orchestrate dynamic kinetochore-microtubule interaction for faithful chromosome segregation. PMID:22110139

NSF- and SNARE-mediated membrane fusion is required for nuclear envelope formation and completion of nuclear pore complex assembly in Xenopus laevis egg extracts.

PubMed

Baur, Tina; Ramadan, Kristijan; Schlundt, Andreas; Kartenbeck, Jürgen; Meyer, Hemmo H

2007-08-15

Despite the progress in understanding nuclear envelope (NE) reformation after mitosis, it has remained unclear what drives the required membrane fusion and how exactly this is coordinated with nuclear pore complex (NPC) assembly. Here, we show that, like other intracellular fusion reactions, NE fusion in Xenopus laevis egg extracts is mediated by SNARE proteins that require activation by NSF. Antibodies against Xenopus NSF, depletion of NSF or the dominant-negative NSF(E329Q) variant specifically inhibited NE formation. Staging experiments further revealed that NSF was required until sealing of the envelope was completed. Moreover, excess exogenous alpha-SNAP that blocks SNARE function prevented membrane fusion and caused accumulation of non-flattened vesicles on the chromatin surface. Under these conditions, the nucleoporins Nup107 and gp210 were fully recruited, whereas assembly of FxFG-repeat-containing nucleoporins was blocked. Together, we define NSF- and SNARE-mediated membrane fusion events as essential steps during NE formation downstream of Nup107 recruitment, and upstream of membrane flattening and completion of NPC assembly.

Xenopus: An Emerging Model for Studying Congenital Heart Disease

PubMed Central

Kaltenbrun, Erin; Tandon, Panna; Amin, Nirav M.; Waldron, Lauren; Showell, Chris; Conlon, Frank L.

2011-01-01

Congenital heart defects affect nearly 1% of all newborns and are a significant cause of infant death. Clinical studies have identified a number of congenital heart syndromes associated with mutations in genes that are involved in the complex process of cardiogenesis. The African clawed frog, Xenopus, has been instrumental in studies of vertebrate heart development and provides a valuable tool to investigate the molecular mechanisms underlying human congenital heart diseases. In this review, we discuss the methodologies that make Xenopus an ideal model system to investigate heart development and disease. We also outline congenital heart conditions linked to cardiac genes that have been well-studied in Xenopus and describe some emerging technologies that will further aid in the study of these complex syndromes. PMID:21538812

Scythe regulates apoptosis through modulating ubiquitin-mediated proteolysis of the Xenopus elongation factor XEF1AO

PubMed Central

Minami, Ryosuke; Shimada, Masumi; Yokosawa, Hideyoshi; Kawahara, Hiroyuki

2007-01-01

Scythe was originally identified as a novel Reaper-binding anti-apoptotic protein, although the mechanisms of its functions remain largely obscure. Our previous analysis revealed that Scythe can bind to a proteasomal subunit via N-terminal domains and that the domains are required for appropriate development of Xenopus embryos. In the present study, we show evidence that the N-terminus of Scythe interacts with XEF1AO, a maternal form of Xenopus laevis EF1A that was suggested to be a potential inducer of apoptosis in vertebrates, and that the binding enhances the poly-ubiquitin modification and subsequent degradation of XEF1AO. Scythe is required for degradation of XEF1AO, since immunodepletion of Scythe from embryonic extracts stabilized XEF1AO significantly. Furthermore, we show that apoptosis induced by accumulation of XEF1AO can be suppressed by co-expression of the full-length form of Scythe. These observations indicate that the proteolytic regulation of XEF1AO, mediated through Scythe, is essential to prevent inappropriate accumulation of XEF1AO and resulting apoptotic events during the course of Xenopus development. PMID:17428197

Protein kinase and phosphatase modulation of quail brain GABA(A) and non-NMDA receptors co-expressed in Xenopus oocytes.

PubMed

Moon, C; Fraser, S P; Djamgoz, M B

2000-02-01

The GABA(A) receptor and the non-NMDA subtype of the ionotropic glutamate receptor were co-expressed in Xenopus oocytes by injection of quail brain mRNA. The oocytes were treated with various protein kinase (PK) and protein phosphatase (PP) activators and inhibitors and the effects on receptor functioning were monitored. Two phorbol esters, 4-beta-phorbol 12-myristate-13-acetate (PMA) and 4-beta-phorbol 12,13-dibutyrate (PDBu); the cGMP-dependent PK activators sodium nitroprusside (SNP) and S-nitrosoglutathione (SNOG); and the PP inhibitor okadaic acid (OA) reduced the amplitude of the GABA-induced currents, whilst the PK inhibitor staurosporine potentiated it. In addition, PMA, PDBu, SNP, and OA reduced the desensitization of the GABA-induced response. Identical treatments generally had similar but less pronounced effects on responses generated by kainate (KA) but the desensitization characteristic of the non-NMDA receptor was not affected. None of the treatments had any effect on the reversal potentials of the induced currents. Immunoblots revealed that the oocytes express endogenous PKG and guanylate cyclase. The results are discussed in terms of the molecular structures of GABA(A) and non-NMDA receptors and the potential functional consequences of phosphorylation/dephosphorylation.

Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS).

PubMed

Lombard-Banek, Camille; Reddy, Sushma; Moody, Sally A; Nemes, Peter

2016-08-01

Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ∼75-amol (∼11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or <0.2% of the total protein contained in a blastomere in the 16-cell embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The unexpected teratogenicity of RXR antagonist UVI3003 via activation of PPARγ in Xenopus tropicalis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Jingmin

2017-01-01

The RXR agonist (triphenyltin, TPT) and the RXR antagonist (UVI3003) both show teratogenicity and, unexpectedly, induce similar malformations in Xenopus tropicalis embryos. In the present study, we exposed X. tropicalis embryos to UVI3003 in seven specific developmental windows and identified changes in gene expression. We further measured the ability of UVI3003 to activate Xenopus RXRα (xRXRα) and PPARγ (xPPARγ) in vitro and in vivo. We found that UVI3003 activated xPPARγ either in Cos7 cells (in vitro) or Xenopus embryos (in vivo). UVI3003 did not significantly activate human or mouse PPARγ in vitro; therefore, the activation of Xenopus PPARγ by UVI3003more » is novel. The ability of UVI3003 to activate xPPARγ explains why UVI3003 and TPT yield similar phenotypes in Xenopus embryos. Our results indicate that activating PPARγ leads to teratogenic effects in Xenopus embryos. More generally, we infer that chemicals known to specifically modulate mammalian nuclear hormone receptors cannot be assumed to have the same activity in non-mammalian species, such as Xenopus. Rather they must be tested for activity and specificity on receptors of the species in question to avoid making inappropriate conclusions. - Highlights: • UVI3003 is a RXRs antagonist and shows teratogenicity to Xenopus embryos. • UVI3003 activated xPPARγ either in Cos7 cells or Xenopus embryos. • UVI3003 did not activate human or mouse PPARγ in Cos7 cells. • Activating PPARγ leads to teratogenic effects in Xenopus embryos.« less

A highly efficient, cell-free translation/translocation system prepared from Xenopus eggs.

PubMed Central

Matthews, G; Colman, A

1991-01-01

We describe the use of a Xenopus laevis egg extract for the in vitro translation and post translational modification of membrane and secretory proteins. This extract is capable of the translation and segregation into membranes of microgram per millilitre levels of protein from added mRNAs. Signal sequences of segregated proteins are efficiently cleaved and appropriate N-linked glycosylation patterns are produced. The extract also supports the quantitative assembly of murine immunoglobulin heavy and light chains into tetramers, and two events which take place beyond the endoplasmic reticulum, mannose 6 phosphorylation of murine cathepsin D and O-linked glycosylation of coronavirus E1 protein, also occur, but at reduced efficiency. The stability of the membranes allows protease protection studies and quantitative centrifugal fractionation of segregated and unsegregated proteins to be performed. Conditions for the use of stored extract have also been determined. Images PMID:1754376

Mitotic trigger waves and the spatial coordination of the Xenopus cell cycle.

PubMed

Chang, Jeremy B; Ferrell, James E

2013-08-29

Despite the large size of the Xenopus laevis egg (approximately 1.2 mm diameter), a fertilized egg rapidly proceeds through mitosis in a spatially coordinated fashion. Mitosis is initiated by a bistable system of regulatory proteins centred on Cdk1 (refs 1, 2), raising the possibility that this spatial coordination could be achieved through trigger waves of Cdk1 activity. Using an extract system that performs cell cycles in vitro, here we show that mitosis does spread through Xenopus cytoplasm via trigger waves, propagating at a linear speed of approximately 60 µm min(-1). Perturbing the feedback loops that give rise to the bistability of Cdk1 changes the speed and dynamics of the waves. Time-lapse imaging of intact eggs argues that trigger waves of Cdk1 activation are responsible for surface contraction waves, ripples in the cell cortex that precede cytokinesis. These findings indicate that Cdk1 trigger waves help ensure the spatiotemporal coordination of mitosis in large eggs. Trigger waves may be an important general mechanism for coordinating biochemical events over large distances.

Adult amphibian epidermal proteins: biochemical characterization and developmental appearance.

PubMed

Reeves, O R

1975-08-01

The keratin-like proteins (KLPs) from the epidermis of adult frogs of the species Xenopus laevis have been isolated and biochemically characterized by means of polyacrylamide gel electrophoresis, amino acid analysis, tryptic peptide mapping, amino-terminal end-group analysis and isoelectric focusing. One particular protein fraction of rather unusual amino acid composition found only in epidermal tissue was isolated in quantity by preparative gel electrophoresis and monospecific antibodies prepared against it. Using this anti-KLP antibody preparation it was possible to show that at least one kine of keratin-like protein characteristic of the adult epidermis first appears within the larval epidermis during metamorphosis. This is the first reported biochemical characterization of a tissue-specific protien from adult amphibian skin.

Identification of Proteins Required for Precise Positioning of Apc2 in Dendrites

PubMed Central

Weiner, Alexis T.; Seebold, Dylan Y.; Michael, Nick L.; Guignet, Michelle; Feng, Chengye; Follick, Brandon; Yusko, Brandon A.; Wasilko, Nathan P.; Torres-Gutierrez, Pedro; Rolls, Melissa M.

2018-01-01

In Drosophila neurons, uniform minus-end-out polarity in dendrites is maintained in part by kinesin-2-mediated steering of growing microtubules at branch points. Apc links the kinesin motor to growing microtubule plus ends and Apc2 recruits Apc to branch points where it functions. Because Apc2 acts to concentrate other steering proteins to branch points, we wished to understand how Apc2 is targeted. From an initial broad candidate RNAi screen, we found Miro (a mitochondrial transport protein), Ank2, Axin, spastin and Rac1 were required to position Apc2-GFP at dendrite branch points. YFP-Ank2-L8, Axin-GFP and mitochondria also localized to branch points suggesting the screen identified relevant proteins. By performing secondary screens, we found that energy production by mitochondria was key for Apc2-GFP positioning and spastin acted upstream of mitochondria. Ank2 seems to act independently from other players, except its membrane partner, Neuroglian (Nrg). Rac1 likely acts through Arp2/3 to generate branched actin to help recruit Apc2-GFP. Axin can function in a variety of wnt signaling pathways, one of which includes heterotrimeric G proteins and Frizzleds. Knockdown of Gαs, Gαo, Fz and Fz2, reduced targeting of Apc2 and Axin to branch points. Overall our data suggest that mitochondrial energy production, Nrg/Ank2, branched actin generated by Arp2/3 and Fz/G proteins/Axin function as four modules that control localization of the microtubule regulator Apc2 to its site of action in dendrite branch points. PMID:29602811

Loss of function of Saccharomyces cerevisiae kinesin-related CIN8 and KIP1 is suppressed by KAR3 motor domain mutations.

PubMed

Hoyt, M A; He, L; Totis, L; Saunders, W S

1993-09-01

The kinesin-related products of the CIN8 and KIP1 genes of Saccharomyces cerevisiae redundantly perform an essential function in mitosis. The action of either gene-product is required for an outwardly directed force that acts upon the spindle poles. We have selected mutations that suppress the temperature-sensitivity of a cin8-temperature-sensitive kip1-delta strain. The extragenic suppressors analyzed were all found to be alleles of the KAR3 gene. KAR3 encodes a distinct kinesin-related protein whose action antagonizes Cin8p/Kip1p function. All seven alleles analyzed were altered within the region of KAR3 that encodes the putative force-generating (or "motor") domain. These mutations also suppressed the inviability associated with the cin8-delta kip1-delta genotype, a property not shared by a deletion of KAR3. Other properties of the suppressing alleles revealed that they were not null for function. Six of the seven were unaffected for the essential karyogamy and meiosis properties of KAR3 and the seventh was dominant for the suppressing trait. Our findings suggest that despite an antagonistic relationship between Cin8p/Kip1p and Kar3p, aspects of their mitotic roles may be similar.

Gene structure, transcripts and calciotropic effects of the PTH family of peptides in Xenopus and chicken.

PubMed

Pinheiro, Pedro L C; Cardoso, João C R; Gomes, Ana S; Fuentes, Juan; Power, Deborah M; Canário, Adelino V M

2010-12-01

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) belong to a family of endocrine factors that share a highly conserved N-terminal region (amino acids 1-34) and play key roles in calcium homeostasis, bone formation and skeletal development. Recently, PTH-like peptide (PTH-L) was identified in teleost fish raising questions about the evolution of these proteins. Although PTH and PTHrP have been intensively studied in mammals their function in other vertebrates is poorly documented. Amphibians and birds occupy unique phylogenetic positions, the former at the transition of aquatic to terrestrial life and the latter at the transition to homeothermy. Moreover, both organisms have characteristics indicative of a complex system in calcium regulation. This study investigated PTH family evolution in vertebrates with special emphasis on Xenopus and chicken. The PTH-L gene is present throughout the vertebrates with the exception of placental mammals. Gene structure of PTH and PTH-L seems to be conserved in vertebrates while PTHrP gene structure is divergent and has acquired new exons and alternative promoters. Splice variants of PTHrP and PTH-L are common in Xenopus and chicken and transcripts of the former have a widespread tissue distribution, although PTH-L is more restricted. PTH is widely expressed in fish tissue but from Xenopus to mammals becomes largely restricted to the parathyroid gland. The N-terminal (1-34) region of PTH, PTHrP and PTH-L in Xenopus and chicken share high sequence conservation and the capacity to modify calcium fluxes across epithelia suggesting a conserved role in calcium metabolism possibly via similar receptors. The parathyroid hormone family contains 3 principal members, PTH, PTHrP and the recently identified PTH-L. In teleosts there are 5 genes which encode PTHrP (2), PTH (2) and PTH-L and in tetrapods there are 3 genes (PTHrP, PTH and PTH-L), the exception is placental mammals which have 2 genes and lack PTH-L. It is hypothesized that

Light responses in rods of vitamin A-deprived Xenopus.

PubMed

Solessio, Eduardo; Umino, Yumiko; Cameron, David A; Loew, Ellis; Engbretson, Gustav A; Knox, Barry E; Barlow, Robert B

2009-09-01

Accumulation of free opsin by mutations in rhodopsin or insufficiencies in the visual cycle can lead to retinal degeneration. Free opsin activates phototransduction; however, the link between constitutive activation and retinal degeneration is unclear. In this study, the photoresponses of Xenopus rods rendered constitutively active by vitamin A deprivation were examined. Unlike their mammalian counterparts, Xenopus rods do not degenerate. Contrasting phototransduction in vitamin A-deprived Xenopus rods with phototransduction in constitutively active mammalian rods may provide new understanding of the mechanisms that lead to retinal degeneration. The photocurrents of Xenopus tadpole rods were measured with suction electrode recordings, and guanylate cyclase activity was measured with the IBMX (3-isobutyl-1-methylxanthine) jump technique. The amount of rhodopsin in rods was determined by microspectrophotometry. The vitamin A-deprived rod outer segments were 60% to 70% the length and diameter of the rods in age-matched animals. Approximately 90% of its opsin content was in the free or unbound form. Analogous to bleaching adaptation, the photoresponses were desensitized (10- to 20-fold) and faster. Unlike bleaching adaptation, the vitamin A-deprived rods maintained near normal saturating (dark) current densities by developing abnormally high rates of cGMP synthesis. Their rate of cGMP synthesis in the dark (15 seconds(-1)) was twofold greater than the maximum levels attainable by control rods ( approximately 7 seconds(-1)). Preserving circulating current density and response range appears to be an important goal for rod homeostasis. However, the compensatory changes associated with vitamin A deprivation in Xenopus rods come at the high metabolic cost of a 15-fold increase in basal ATP consumption.

Global gene expression during early differentiation of Xenopus (Silurana) tropicalis gonad tissues

EPA Science Inventory

African clawed frog Xenopus sp. has been used extensively for developmental biology and toxicology research. Xenopus (Silurana) tropicalis has been coveted more recently for genomics research because its diploid genome has been sequenced. Amid concerns of environmental pollutants...

Inference of genetic network of Xenopus frog egg: improved genetic algorithm.

PubMed

Wu, Shinq-Jen; Chou, Chia-Hsien; Wu, Cheng-Tao; Lee, Tsu-Tian

2006-01-01

An improved genetic algorithm (IGA) is proposed to achieve S-system gene network modeling of Xenopus frog egg. Via the time-courses training datasets from Michaelis-Menten model, the optimal parameters are learned. The S-system can clearly describe activative and inhibitory interaction between genes as generating and consuming process. We concern the mitotic control in cell-cycle of Xenopus frog egg to realize cyclin-Cdc2 and Cdc25 for MPF activity. The proposed IGA can achieve global search with migration and keep the best chromosome with elitism operation. The generated gene regulatory networks can provide biological researchers for further experiments in Xenopus frog egg cell cycle control.

HIP1-ALK, a novel fusion protein identified in lung adenocarcinoma.

PubMed

Hong, Mineui; Kim, Ryong Nam; Song, Ji-Young; Choi, So-Jung; Oh, Ensel; Lira, Maruja E; Mao, Mao; Takeuchi, Kengo; Han, Joungho; Kim, Jhingook; Choi, Yoon-La

2014-03-01

The most common mechanism underlying overexpression and activation of anaplastic lymphoma kinase (ALK) in non-small-cell lung carcinoma could be attributed to the formation of a fusion protein. To date, five fusion partners of ALK have been reported, namely, echinoderm microtubule associated protein like 4, tropomyosin-related kinase-fused gene, kinesin family member 5B, kinesin light chain 1, and protein tyrosine phosphatase, nonreceptor type 3. In this article, we report a novel fusion gene huntingtin interacting protein 1 (HIP1)-ALK, which is conjoined between the huntingtin-interacting protein 1 gene HIP1 and ALK. Reverse-transcriptase polymerase chain reaction and immunohistochemical analysis were used to detect this fusion gene's transcript and protein expression, respectively. We had amplified the full-length cDNA sequence of this novel fusion gene by using 5'-rapid amplification of cDNA ends. The causative genomic translocation t(2;7)(p23;q11.23) for generating this novel fusion gene was verified by using genomic sequencing. The examined adenocarcinoma showed predominant acinar pattern, and ALK immunostaining was localized to the cytoplasm, with intense staining in the submembrane region. In break-apart, fluorescence in situ hybridization analysis for ALK, split of the 5' and 3' probe signals, and isolated 3' signals were observed. Reverse-transcriptase polymerase chain reaction revealed that the tumor harbored a novel fusion transcript in which exon 21 of HIP1 was fused to exon 20 of ALK in-frame. The novel fusion gene and its protein HIP1-ALK harboring epsin N-terminal homology, coiled-coil, juxtamembrane, and kinase domains, which could play a role in carcinogenesis, could become diagnostic and therapeutic target of the lung adenocarcinoma and deserve a further study in the future.

Transforming Growth Factor Beta (TGFβ) Is Produced by and Influences the Proliferative Response of Xenopus laevis Lymphocytes

PubMed Central

Haynes, Laura

1993-01-01

Both TGF/β2 and 5 have been described in the South African clawed frog Xenopus laevis and have been cloned from the tadpole-derived fibroblast cell line, XTC. Because TGFβ has such a profound inhibitory effect on the mammalian immune system, this study was performed to determine whether TGFβ: (a) has any in vitro effects on the growth of Xenopus lymphoblasts, and (b) is produced by mitogen-activated Xenopus lymphocytes. Following stimulation with mitogen or alloantigen, T lymphocytes from Xenopus secrete a T-cell growth factor (TCGF) that is functionally homologous to mammalian interleukin-2 (IL-2). Both recombinant human TGFβ1 and Xenopus TGFβ5 inhibit TCGF-induced proliferation of Xenopus splenic blasts and this inhibition can be reversed with anti-pan TGFβ antiserum. The Xenopus mitogen-induced saturated ammonium sulfate precipitated TCGF-containing supernatant (SAS TCGF SN) also contains latent TGFβ as assayed on mink lung fibroblasts and Xenopus splenic blasts, and experiments utilizing anti-TGFβ antiserum showed that only TGFβ5 is present in this supernatant. PMID:8281035

Live imaging of targeted cell ablation in Xenopus: a new model to study demyelination and repair

PubMed Central

Kaya, F.; Mannioui, A.; Chesneau, A.; Sekizar, S.; Maillard, E.; Ballagny, C.; Houel-Renault, L.; Du Pasquier, D.; Bronchain, O.; Holtzmann, I.; Desmazieres, A.; Thomas, J.-L.; Demeneix, B. A.; Brophy, P. J.; Zalc, B.; Mazabraud, A.

2012-01-01

Live imaging studies of the processes of demyelination and remyelination have so far been technically limited in mammals. We have thus generated a Xenopus laevis transgenic line allowing live imaging and conditional ablation of myelinating oligodendrocytes throughout the central nervous system (CNS). In these transgenic pMBP-eGFP-NTR tadpoles the myelin basic protein (MBP) regulatory sequences, specific to mature oligodendrocytes, are used to drive expression of an eGFP (enhanced green fluorescent protein) reporter fused to the E. coli nitroreductase (NTR) selection enzyme. This enzyme converts the innocuous pro-drug metronidazole (MTZ) to a cytotoxin. Using two-photon imaging in vivo, we show that pMBP-eGFP-NTR tadpoles display a graded oligodendrocyte ablation in response to MTZ, which depends on the exposure time to MTZ. MTZ-induced cell death was restricted to oligodendrocytes, without detectable axonal damage. After cessation of MTZ treatment, remyelination proceeded spontaneously, but was strongly accelerated by retinoic acid. Altogether, these features establish the Xenopus pMBP-eGFP-NTR line as a novel in vivo model for the study of demyelination/remyelination processes and for large-scale screens of therapeutic agents promoting myelin repair. PMID:22973012

Probing the Xenopus laevis inner ear transcriptome for biological function

PubMed Central

2012-01-01

Background The senses of hearing and balance depend upon mechanoreception, a process that originates in the inner ear and shares features across species. Amphibians have been widely used for physiological studies of mechanotransduction by sensory hair cells. In contrast, much less is known of the genetic basis of auditory and vestibular function in this class of animals. Among amphibians, the genus Xenopus is a well-characterized genetic and developmental model that offers unique opportunities for inner ear research because of the amphibian capacity for tissue and organ regeneration. For these reasons, we implemented a functional genomics approach as a means to undertake a large-scale analysis of the Xenopus laevis inner ear transcriptome through microarray analysis. Results Microarray analysis uncovered genes within the X. laevis inner ear transcriptome associated with inner ear function and impairment in other organisms, thereby supporting the inclusion of Xenopus in cross-species genetic studies of the inner ear. The use of gene categories (inner ear tissue; deafness; ion channels; ion transporters; transcription factors) facilitated the assignment of functional significance to probe set identifiers. We enhanced the biological relevance of our microarray data by using a variety of curation approaches to increase the annotation of the Affymetrix GeneChip® Xenopus laevis Genome array. In addition, annotation analysis revealed the prevalence of inner ear transcripts represented by probe set identifiers that lack functional characterization. Conclusions We identified an abundance of targets for genetic analysis of auditory and vestibular function. The orthologues to human genes with known inner ear function and the highly expressed transcripts that lack annotation are particularly interesting candidates for future analyses. We used informatics approaches to impart biologically relevant information to the Xenopus inner ear transcriptome, thereby addressing the

Identification of PDC-109-like protein(s) in buffalo seminal plasma.

PubMed

Harshan, Hiron M; Sankar, Surya; Singh, L P; Singh, Manish Kumar; Sudharani, S; Ansari, M R; Singh, S K; Majumdar, A C; Joshi, P

2009-10-01

The FN-2 family of seminal plasma proteins represents the major protein fraction of bovine seminal plasma. These proteins also constitute the major seminal plasma proteins fraction in horse, goat and bison seminal plasma and are present in pig, rat, mouse, hamster and human seminal plasma. BSP-A1 and BSP-A2, the predominant proteins of the FN-2 family, are collectively termed as PDC-109. Fn-2 proteins play an important role in fertilization, including sperm capacitation and formation of oviductal sperm reservoirs. Significantly, BSP proteins were also shown to have negative effects in the context of sperm storage. No conclusive evidence for the presence of buffalo seminal plasma protein(s) similar to PDC-109 exists. Studies with buffalo seminal plasma indicated that isolation and identification of PDC-109-like protein(s) from buffalo seminal plasma by conventional methods might be difficult. Thus, antibodies raised against PDC-109 isolated, and purified from cattle seminal plasma, were used for investigating the presence of PDC-109-like protein(s) in buffalo seminal plasma. Buffalo seminal plasma proteins were resolved on SDS-PAGE, blotted to nitro cellulose membranes and probed for the presence of PDC-109-like protein(s) using the PDC-109 antisera raised in rabbits. A distinct immunoreactive band well below the 20-kDa regions indicated the presence of PDC-109-like protein(s) in buffalo seminal plasma.

Human kidney anion exchanger 1 interacts with kinesin family member 3B (KIF3B)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duangtum, Natapol; Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700; Junking, Mutita

Highlights: {yields} Impaired trafficking of kAE1 causes distal renal tubular acidosis (dRTA). {yields} The interaction between kAE1 and kinesin family member 3B (KIF3B) is reported. {yields} The co-localization between kAE and KIF3B was detected in human kidney tissues. {yields} A marked reduction of kAE1 on the cell membrane was observed when KIF3B was knockdown. {yields} KFI3B plays an important role in trafficking of kAE1 to the plasma membrane. -- Abstract: Impaired trafficking of human kidney anion exchanger 1 (kAE1) to the basolateral membrane of {alpha}-intercalated cells of the kidney collecting duct leads to the defect of the Cl{sup -}/HCO{sub 3}{supmore » -} exchange and the failure of proton (H{sup +}) secretion at the apical membrane of these cells, causing distal renal tubular acidosis (dRTA). In the sorting process, kAE1 interacts with AP-1 mu1A, a subunit of AP-1A adaptor complex. However, it is not known whether kAE1 interacts with motor proteins in its trafficking process to the plasma membrane or not. We report here that kAE1 interacts with kinesin family member 3B (KIF3B) in kidney cells and a dileucine motif at the carboxyl terminus of kAE1 contributes to this interaction. We have also demonstrated that kAE1 co-localizes with KIF3B in human kidney tissues and the suppression of endogenous KIF3B in HEK293T cells by small interfering RNA (siRNA) decreases membrane localization of kAE1 but increases its intracellular accumulation. All results suggest that KIF3B is involved in the trafficking of kAE1 to the plasma membrane of human kidney {alpha}-intercalated cells.« less

Survey of the vestibulum, and behavior of Xenopus laevis larvae developed during a 7-days space flight.

PubMed

Briegleb, W; Neubert, J; Schatz, A; Klein, T; Kruse, B

1986-01-01

Aquatic animals have almost no body weight related proprioception for spatial orientation. Xenopus larvae, like fish, maintain their attitude in water by continuous correction with their fin(s). For these reasons a special performance of the equilibrium system compared to terrestrial animals is necessary. Evidently fish therefore have more compact (dense) otoliths; Xenopus larvae have less dense otolith (membranes) similar to land vertebrates; but their sacculus-otoliths are vertically positioned, which also may lead to a higher g-sensitivity. For plausibility reasons gravity should influence the embryonic development of gravity receptors. Yet, evaluations of photographs taken from the surface of cut deep-frozen objects by incident light show no aberration of the shape of the whole vestibulum and of the shape, density, size and position of the otolith membrane in larvae developed under near-zero g (NEXPA-BW-STATEX in D-1-Mission). The further evaluation of the "weightless-larvae" revealed a probably not yet described statolith-like formation in the dorsal wall of the vestibulum. In the weightless larvae this formation outnumbers, also qualitatively, strongly the l-g controls. An extra result is the lack of striking effects of cosmic radiation on the embryonic development of the flown Xenopus eggs. The swimming behavior of the larvae which was observed about one hour after landing of the Space Shuttle showed a typical anomaly (loop swimming), which is known from larvae developed on the clinostat or from fish flown aboard Apollo capsules.

Survey of the vestibulum, and behavior of xenopus laevis larvae developed during a 7-days space flight

NASA Astrophysics Data System (ADS)

Briegleb, W.; Neubert, J.; Schatz, A.; Klein, T.; Kruse, B.

Aquatic animals have almost no body weight related proprioception for spatial orientation. Xenopus larvae, like fish, maintain their attitude in water by continuous correction with their fin(s). For these reasons a special performance of the equilibrium system compared to terrestrial animals is necessary. Evidently fish therefore have more compact (dense) otoliths; Xenopus larvae have less dense otolith (membranes) similar to land vertebrates; but their sacculus-otoliths are vertically positioned, which also may lead to a higher g-sensitivity. For plausibility reasons gravity should influence the embryonic development of gravity receptors. Yet, evaluations of photographs taken from the surface of cut deep-frozen objects by incident light show no aberration of the shape of the whole vestibulum and of the shape, density, size and position of the otolith membrane in larvae developed under near-zero g (NEXPA-BW-STATEX in D1-Mission). The further evaluation of the ``weightless-larvae'' revealed a probably not yet described statolith-like formation in the dorsal wall of the vestibulum. In the weightless larvae this formation outnumbers, also qualitatively, strongly the 1-g controls. An extra result is the lack of striking effects of cosmic radiation on the embryonic development of the flown Xenopus eggs. The swimming behavior of the larvae which was observed about one hour after landing of the Space Shuttle showed a typical anomaly (loop swimming), which is known from larvae developed on the clinostat or from fish flown aboard Apollo capsules.

Identification of Proteins Required for Precise Positioning of Apc2 in Dendrites.

PubMed

Weiner, Alexis T; Seebold, Dylan Y; Michael, Nick L; Guignet, Michelle; Feng, Chengye; Follick, Brandon; Yusko, Brandon A; Wasilko, Nathan P; Torres-Gutierrez, Pedro; Rolls, Melissa M

2018-05-04

In Drosophila neurons, uniform minus-end-out polarity in dendrites is maintained in part by kinesin-2-mediated steering of growing microtubules at branch points. Apc links the kinesin motor to growing microtubule plus ends and Apc2 recruits Apc to branch points where it functions. Because Apc2 acts to concentrate other steering proteins to branch points, we wished to understand how Apc2 is targeted. From an initial broad candidate RNAi screen, we found Miro (a mitochondrial transport protein), Ank2, Axin, spastin and Rac1 were required to position Apc2-GFP at dendrite branch points. YFP-Ank2-L8, Axin-GFP and mitochondria also localized to branch points suggesting the screen identified relevant proteins. By performing secondary screens, we found that energy production by mitochondria was key for Apc2-GFP positioning and spastin acted upstream of mitochondria. Ank2 seems to act independently from other players, except its membrane partner, Neuroglian (Nrg). Rac1 likely acts through Arp2/3 to generate branched actin to help recruit Apc2-GFP. Axin can function in a variety of wnt signaling pathways, one of which includes heterotrimeric G proteins and Frizzleds. Knockdown of Gαs, Gαo, Fz and Fz2, reduced targeting of Apc2 and Axin to branch points. Overall our data suggest that mitochondrial energy production, Nrg/Ank2, branched actin generated by Arp2/3 and Fz/G proteins/Axin function as four modules that control localization of the microtubule regulator Apc2 to its site of action in dendrite branch points. Copyright © 2018 Weiner et al.

Effects of ketamine on glucose uptake by glucose transporter type 3 expressed in Xenopus oocytes: The role of protein kinase C

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomioka, Shigemasa, E-mail: tomioka@dent.tokushima-u.ac.jp; Kaneko, Miyuki; Satomura, Kazuhito

2009-10-09

We investigated the effects of ketamine on the type 3 facilitative glucose transporter (GLUT3), which plays a major role in glucose transport across the plasma membrane of neurons. Human-cloned GLUT3 was expressed in Xenopus oocytes by injection of GLUT3 mRNA. GLUT3-mediated glucose uptake was examined by measuring oocyte radioactivity following incubation with 2-deoxy-D-[1,2-{sup 3}H]glucose. While ketamine and S(+)-ketamine significantly increased GLUT3-mediated glucose uptake, this effect was biphasic such that higher concentrations of ketamine inhibited glucose uptake. Ketamine (10 {mu}M) significantly increased V{sub max} but not K{sub m} of GLUT3 for 2-deoxy-D-glucose. Although staurosporine (a protein kinase C inhibitor) increased glucosemore » uptake, no additive or synergistic interactions were observed between staurosporine and racemic ketamine or S(+)-ketamine. Treatment with ketamine or S(+)-ketamine partially prevented GLUT3 inhibition by the protein kinase C activator phorbol-12-myrisate-13-acetate. Our results indicate that ketamine increases GLUT3 activity at clinically relevant doses through a mechanism involving PKC inhibition.« less

ZWY Sex Determination in Xenopus tropicalis

EPA Science Inventory

Most vertebrate species with described genetic sex determination are either male (XY) or female (ZW) heterogametic. To date, studies with Xenopus species indicate that members of this genus operate under a ZW sex determination system. We used two different approaches and demonst...

Different forms of soluble cytoplasmic mRNA binding proteins and particles in Xenopus laevis oocytes and embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murray, M.T.; Krohne, G.; Franke, W.W.

1991-01-01

To gain insight into the mechanisms involved in the formation of maternally stored mRNPs during Xenopus laevis development, we searched for soluble cytoplasmic proteins of the oocyte that are able to selectively bind mRNAs, using as substrate radiolabeled mRNA. In vitro mRNP assembly in solution was followed by UV-cross-linking and RNase digestion, resulting in covalent tagging of polypeptides by nucleotide transfer. Five polypeptides of approximately 54, 56 60, 70, and 100 kD (p54, p56, p60, p70, and p100) have been found to selectively bind mRNA and assemble into mRNPs. These polypeptides, which correspond to previously described native mRNP components, occurmore » in three different particle classes of approximately 4.5S, approximately 6S, and approximately 15S, as also determined by their reactions with antibodies against p54 and p56. Whereas the approximately 4.5S class contains p42, p60, and p70, probably each in the form of individual molecules or small complexes, the approximately 6S particles appears to consist only of p54 and p56, which occur in a near-stoichiometric ratio suggestive of a heterodimer complex. The approximately 15S particles contain, in addition to p54 and p56, p60 and p100 and this is the single occurring form of RNA-binding p100. We have also observed changes in the in vitro mRNA binding properties of these polypeptides during oogenesis and early embryonic development, in relation to their phosphorylation state and to the activity of an approximately 15S particle-associated protein kinase, suggesting that these proteins are involved in the developmental translational regulation of maternal mRNAs.« less

Variants of the Xenopus laevis ribosomal transcription factor xUBF are developmentally regulated by differential splicing.

PubMed

Guimond, A; Moss, T

1992-07-11

XUBF is a Xenopus ribosomal transcription factor of the HMG-box family which contains five tandemly disposed homologies to the HMG1 & 2 DNA binding domains. XUBF has been isolated as a protein doublet and two cDNAs encoding the two molecular weight variants have been characterised. The major two forms of xUBF identified differ by the presence or absence of a 22 amino acid segment lying between HMG-boxes 3 and 4. Here we show that the mRNAs for these two forms of xUBF are regulated during development and differentiation over a range of nearly 20 fold. By isolating two of the xUBF genes, it was possible to show that both encoded the variable 22 amino acid segment in exon 12. Oocyte splicing assays and the sequencing of PCR-generated cDNA fragments, demonstrated that the transcripts from one of these genes were differentially spliced in a developmentally regulated manner. Transcripts from the second gene were found to be predominantly or exclusively spliced to produce the lower molecular weight form of xUBF. Expression of a high molecular weight form from yet a third gene was also detected. Although the intron-exon structures of the Xenopus and mouse UBF genes were found to be essentially identical, the differential splicing of exon 8 found in mammals, was not detected in Xenopus.

cis- and trans-acting elements of the estrogen-regulated vitellogenin gene B1 of Xenopus laevis.

PubMed

Wahli, W; Martinez, E; Corthésy, B; Cardinaux, J R

1989-01-01

Vitellogenin genes are expressed under strict estrogen control in the liver of female oviparous vertebrates. Gene transfer experiments using estrogen-responsive cells have shown that the 13 bp perfect palindromic element GGTCACTGTGACC found upstream of the Xenopus laevis vitellogenin gene A2 promoter mediates hormonal stimulation and thus, was called the estrogen-responsive element (ERE). In the Xenopus vitellogenin genes B1 and B2 there are two closely adjacent EREs with one or more base substitutions when compared to the consensus ERE GGTCANNNTGACC. On their own, these degenerated elements have only a low or no regulatory capacity at all but act together synergistically to form an estrogen-responsive unit (ERU) with the same strength as the perfect palindromic 13 bp element. Analysis of estrogen receptor binding to the gene B1 ERU revealed a cooperative interaction of receptor dimers to the two adjacent imperfect EREs which most likely explains the synergistic stimulation observed in vivo. Furthermore, a promoter activator element located between positions --113 and --42 of the gene B1 and functional in the human MCF-7 and the Xenopus B3.2 cells has been identified and shown to be involved in the high level of induced transcription activity when the ERE is placed at a distance from the promoter. Finally, a hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to characterize two additional novel cis-acting elements within the vitellogenin gene B1 promoter. One of them, a negative regulatory element (NRE), is responsible for repression of promoter activity in the absence of hormone. The second is related to the NF-I binding site and is required, together with the ERE, to mediate hormonal induction. Moreover, we detected three trans-acting activities in Xenopus liver nuclear extracts that interact with these regions and demonstrated that they participate in the regulation of the expression of the vitellogenin

Control of IP3-mediated Ca2+ puffs in Xenopus laevis oocytes by the Ca2+-binding protein parvalbumin

PubMed Central

John, Linu M; Mosquera-Caro, Monica; Camacho, Patricia; Lechleiter, James D

2001-01-01

Elementary events of Ca2+ release (Ca2+ puffs) can be elicited from discrete clusters of inositol 1,4,5 trisphosphate receptors (IP3Rs) at low concentrations of IP3. Ca2+ puffs have rarely been observed unless elicited by either hormone treatment or introduction of IP3 into the cell. However, cells appear to have sufficient concentrations of IP3 (0.1-3.0 μM) to induce Ca2+ release under resting conditions. Here, we investigated Ca2+ puff activity in non-stimulated Xenopus oocytes using confocal microscopy. The fluorescent Ca2+ dye indicators Calcium Green 1 and Oregon Green 488 BAPTA-2 were injected into oocytes to monitor basal Ca2+ activity. In this preparation, injection or overexpression of parvalbumin, an EF-hand Ca2+-binding protein (CaBP), induced Ca2+ puffs in resting Xenopus oocytes. This activity was inhibited by heparin, an IP3R channel blocker, and by mutation of the Ca2+-binding sites in parvalbumin. Ca2+ puff activity was also evoked by injection of low concentrations of the Ca2+ chelator EGTA, but not by calbindin D28k, another member of the EF-hand CaBP superfamily. BAPTA and the Ca2+ indicator dye Oregon Green 488 BAPTA-1 evoked Ca2+ puff activity, while the dextran conjugate of Oregon Green 488 BAPTA-1 did not. These data indicate that a Ca2+ buffer must be mobile in order to increase Ca2+ puff activity. Together, the data indicate that some IP3Rs spontaneously release Ca2+ under resting concentrations of IP3. These elementary Ca2+ events appear to be below the level of detection of current imaging techniques. We suggest that parvalbumin evokes Ca2+ puffs by coordinating the activity of elementary IP3R channel openings. We conclude that Ca2+ release can be evoked not only by hormone-induced increases in IP3, but also by expression of mobile cytosolic CaBPs under resting concentrations of IP3. PMID:11507154

TPX2 expression is associated with poor survival in gastric cancer.

PubMed

Tomii, Chiharu; Inokuchi, Mikito; Takagi, Yoko; Ishikawa, Toshiaki; Otsuki, Sho; Uetake, Hiroyuki; Kojima, Kazuyuki; Kawano, Tatsuyuki

2017-01-09

Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a microtubule-associated protein required for microtubule formation in human cells. Several studies have demonstrated that TPX2 is overexpressed in multiple tumor types and promotes tumor growth and metastasis. However, there have been few reports regarding its role in gastric cancer. In this study, we evaluated TPX2 expression and investigated its correlations with gastric cancer clinicopathological features and prognosis. Tumor samples were obtained from 290 patients with gastric adenocarcinoma who had undergone gastrectomy. The expression of TPX2 protein was examined using immunohistochemical staining. TPX2 messenger RNA (mRNA) levels were evaluated using real-time quantitative reverse transcription PCR in 19 of the gastric cancer tumors and adjacent normal tissues. The mRNA levels of TPX2 were significantly higher in gastric cancer tissues than in matched adjacent normal tissues (p = 0.004). In the immunohistochemical analysis, TPX2 overexpression was found in 123 (42.4%) of 290 patients. High TPX2 expression was positively associated with age, type of histology, depth of tumor, lymph node metastasis, stage, and remote metastasis or recurrence. High TPX2 expression was significantly associated with poorer disease-specific survival (p = 0.004) and relapse-free interval (p = 0.013). Our results indicated that high TPX2 expression was associated with tumor progression and poor survival in gastric cancer.

Probing Mitotic CENP-E Kinesin with the Tethered Cargo Motion Assay and Laser Tweezers.

PubMed

Gudimchuk, Nikita; Tarasovetc, Ekaterina V; Mustyatsa, Vadim; Drobyshev, Alexei L; Vitre, Benjamin; Cleveland, Don W; Ataullakhanov, Fazly I; Grishchuk, Ekaterina L

2018-06-05

Coiled-coil stalks of various kinesins differ significantly in predicted length and structure; this is an adaption that helps these motors carry out their specialized functions. However, little is known about the dynamic stalk configuration in moving motors. To gain insight into the conformational properties of the transporting motors, we developed a theoretical model to predict Brownian motion of a microbead tethered to the tail of a single, freely walking molecule. This approach, which we call the tethered cargo motion (TCM) assay, provides an accurate measure of the mechanical properties of motor-cargo tethering, verified using kinesin-1 conjugated to a microbead via DNA links in vitro. Applying the TCM assay to the mitotic kinesin CENP-E unexpectedly revealed that when walking along a microtubule track, this highly elongated molecule with a contour length of 230 nm formed a 20-nm-long tether. The stalk of a walking CENP-E could not be extended fully by application of sideways force with optical tweezers (up to 4 pN), implying that CENP-E carries its cargo in a compact configuration. Assisting force applied along the microtubule track accelerates CENP-E walking, but this increase does not depend on the presence of the CENP-E stalk. Our results suggest that the unusually large stalk of CENP-E has little role in regulating its function as a transporter. The adjustable stalk configuration may represent a regulatory mechanism for controlling the physical reach between kinetochore-bound CENP-E and spindle microtubules, or it may assist localizing various kinetochore regulators in the immediate vicinity of the kinetochore-embedded microtubule ends. The TCM assay and underlying theoretical framework will provide a general guide for determining the dynamic configurations of various molecular motors moving along their tracks, freely or under force. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A nuclear factor I-like activity and a liver-specific repressor govern estrogen-regulated in vitro transcription from the Xenopus laevis vitellogenin B1 promoter.

PubMed

Corthésy, B; Cardinaux, J R; Claret, F X; Wahli, W

1989-12-01

A hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to identify novel cis-acting elements within the vitellogenin gene B1 promoter region. In addition to the already well-documented estrogen-responsive element (ERE), two elements were found within the 140 base pairs upstream of the transcription initiation site. One of them, a negative regulatory element, is responsible for the lack of promoter activity in the absence of the hormone and, as demonstrated by DNA-binding assays, interacts with a liver-specific transcription factor. The second is required in association with the estrogen-responsive element to mediate hormonal induction and is recognized by the Xenopus liver homolog of nuclear factor I.

Development of a New Decision Tree to Rapidly Screen Chemical Estrogenic Activities of Xenopus laevis.

PubMed

Wang, Ting; Li, Weiying; Zheng, Xiaofeng; Lin, Zhifen; Kong, Deyang

2014-02-01

During the last past decades, there is an increasing number of studies about estrogenic activities of the environmental pollutants on amphibians and many determination methods have been proposed. However, these determination methods are time-consuming and expensive, and a rapid and simple method to screen and test the chemicals for estrogenic activities to amphibians is therefore imperative. Herein is proposed a new decision tree formulated not only with physicochemical parameters but also a biological parameter that was successfully used to screen estrogenic activities of the chemicals on amphibians. The biological parameter, CDOCKER interaction energy (Ebinding ) between chemicals and the target proteins was calculated based on the method of molecular docking, and it was used to revise the decision tree formulated by Hong only with physicochemical parameters for screening estrogenic activity of chemicals in rat. According to the correlation between Ebinding of rat and Xenopus laevis, a new decision tree for estrogenic activities in Xenopus laevis is finally proposed. Then it was validated by using the randomly 8 chemicals which can be frequently exposed to Xenopus laevis, and the agreement between the results from the new decision tree and the ones from experiments is generally satisfactory. Consequently, the new decision tree can be used to screen the estrogenic activities of the chemicals, and combinational use of the Ebinding and classical physicochemical parameters can greatly improves Hong's decision tree. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression of membrane targeted aequorin in Xenopus laevis oocytes.

PubMed

Daguzan, C; Nicolas, M T; Mazars, C; Leclerc, C; Moreau, M

1995-08-01

We described here a system for high level of expression of the calcium activated photoprotein aequorin. This protein has been targeted to the plasma membrane of Xenopus oocyte by nuclear microinjection of a plasmid containing a construction of a chimeric cDNA encoding a fusion protein composed of the photoprotein aequorin and the 5-HT1A receptor. The expression of this fusion protein is placed under the control of RSV promoter. Functional photoprotein was reconstituted in the oocyte by incubation with coelenterazine. The amount of photoprotein 24 h after nuclear microinjection of the plasmid was sufficient to trigger a detectable light emission following calcium entry. The efficiency of the expression is correlated with the dose of plasmid injected. Intracytoplasmic injection of the plasmid always failed in photoprotein expression. Targeting of the apoprotein was demonstrated by immunolocalization under confocal microscopy. In our experimental conditions, the apoprotein was always localized at the animal pole above the nucleus. We never observed expression and targeting to the plasma membrane of the vegetal pole. WE suggest that such expression might be of great interest for the study of numerous problems of developmental biology, in which calcium-dependent pathways are involved.

Motor domain phosphorylation and regulation of the Drosophila kinesin 13, KLP10A

PubMed Central

Mennella, Vito; Tan, Dong-Yan; Buster, Daniel W.; Asenjo, Ana B.; Rath, Uttama; Ma, Ao; Sosa, Hernando J.

2009-01-01

Microtubule (MT)-destabilizing kinesin 13s perform fundamental roles throughout the cell cycle. In this study, we show that the Drosophila melanogaster kinesin 13, KLP10A, is phosphorylated in vivo at a conserved serine (S573) positioned within the α-helix 5 of the motor domain. In vitro, a phosphomimic KLP10A S573E mutant displays a reduced capacity to depolymerize MTs but normal affinity for the MT lattice. In cells, replacement of endogenous KLP10A with KLP10A S573E dampens MT plus end dynamics throughout the cell cycle, whereas a nonphosphorylatable S573A mutant apparently enhances activity during mitosis. Electron microscopy suggests that KLP10A S573 phosphorylation alters its association with the MT lattice, whereas molecular dynamics simulations reveal how KLP10A phosphorylation can alter the kinesin–MT interface without changing important structural features within the motor’s core. Finally, we identify casein kinase 1α as a possible candidate for KLP10A phosphorylation. We propose a model in which phosphorylation of the KLP10A motor domain provides a regulatory switch controlling the time and place of MT depolymerization. PMID:19687256

JAM related proteins in mucosal homeostasis and inflammation

PubMed Central

Luissint, Anny-Claude; Nusrat, Asma; Parkos, Charles A.

2014-01-01

Mucosal surfaces are lined by epithelial cells that form a physical barrier protecting the body against external noxious substances and pathogens. At a molecular level, the mucosal barrier is regulated by tight junctions (TJs) that seal the paracellular space between adjacent epithelial cells. Transmembrane proteins within TJs include Junctional Adhesion Molecules (JAMs) that belong to the CTX (Cortical Thymocyte marker for Xenopus) family of proteins. JAM family encompasses three classical members (JAM-A, -B and –C) and related molecules including JAM4, JAM-Like protein (JAM-L), Coxsackie and Adenovirus Receptor (CAR), CAR-Like Membrane Protein (CLMP) and Endothelial cell-Selective Adhesion Molecule (ESAM). JAMs have multiple functions that include regulation of endothelial and epithelial paracellular permeability, leukocyte recruitment during inflammation, angiogenesis, cell migration and proliferation. In this review, we summarize the current knowledge regarding the roles of the JAM family members in the regulation of mucosal homeostasis and leukocyte trafficking with a particular emphasis on barrier function and its perturbation during pathological inflammation. PMID:24667924

Asymmetric Distribution of Metals in the Xenopus Laevis Oocyte: a Synchrotron X-Ray Fluorescence Microprobe Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Popescu, B.F.Gh.; Belak, Z.R.; Ignatyev, K.

2009-06-04

The asymmetric distribution of many components of the Xenopus oocyte, including RNA, proteins, and pigment, provides a framework for cellular specialization during development. During maturation, Xenopus oocytes also acquire metals needed for development, but apart from zinc, little is known about their distribution. Synchrotron X-ray fluorescence microprobe was used to map iron, copper, and zinc and the metalloid selenium in a whole oocyte. Iron, zinc, and copper were asymmetrically distributed in the cytoplasm, while selenium and copper were more abundant in the nucleus. A zone of high copper and zinc was seen in the animal pole cytoplasm. Iron was alsomore » concentrated in the animal pole but did not colocalize with zinc, copper, or pigment accumulations. This asymmetry of metal deposition may be important for normal development. Synchrotron X-ray fluorescence microprobe will be a useful tool to examine how metals accumulate and redistribute during fertilization and embryonic development.« less

Asymmetri Distribution of Metals in the Xenopus Laevis Oocyte: a Synchrotron X-Ray Fluorescence Microprobe Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Popescu, B.F.G.; Belak, Z.R.; Ignatyev, K.

2009-04-29

The asymmetric distribution of many components of the Xenopus oocyte, including RNA, proteins, and pigment, provides a framework for cellular specialization during development. During maturation, Xenopus oocytes also acquire metals needed for development, but apart from zinc, little is known about their distribution. Synchrotron X-ray fluorescence microprobe was used to map iron, copper, and zinc and the metalloid selenium in a whole oocyte. Iron, zinc, and copper were asymmetrically distributed in the cytoplasm, while selenium and copper were more abundant in the nucleus. A zone of high copper and zinc was seen in the animal pole cytoplasm. Iron was alsomore » concentrated in the animal pole but did not colocalize with zinc, copper, or pigment accumulations. This asymmetry of metal deposition may be important for normal development. Synchrotron X-ray fluorescence microprobe will be a useful tool to examine how metals accumulate and redistribute during fertilization and embryonic development.« less

Notochord-derived hedgehog is essential for tail regeneration in Xenopus tadpole.

PubMed

Taniguchi, Yuka; Watanabe, Kenji; Mochii, Makoto

2014-06-18

Appendage regeneration in amphibians is regulated by the combinatorial actions of signaling molecules. The requirement of molecules secreted from specific tissues is reflected by the observation that the whole process of regeneration can be inhibited if a certain tissue is removed from the amputated stump. Interestingly, urodeles and anurans show different tissue dependencies during tail regeneration. The spinal cord is essential for tail regeneration in urodele but not in anuran larva, whereas the notochord but not the spinal cord is essential for tail regeneration in anuran tadpoles. Sonic hedgehog is one of the signaling molecules responsible for such phenomenon in axolotl, as hedgehog signaling is essential for overall tail regeneration and sonic hedgehog is exclusively expressed in the spinal cord. In order to know whether hedgehog signaling is involved in the molecular mechanism underlying the inconsistent tissue dependency for tail regeneration between anurans and urodeles, we investigated expression of hedgehog signal-related genes in the regenerating tail of Xenopus tadpole and examined the effect of the hedgehog signal inhibitor, cyclopamine, on the tail regeneration. In Xenopus, sonic hedgehog is expressed exclusively in the notochord but not in the spinal cord of the regenerate. Overall regeneration was severely impaired in cyclopamine-treated tadpoles. Notochord maturation in the regenerate, including cell alignment and vacuolation, and myofiber formation were inhibited. Proliferation of spinal cord cells in the neural ampulla and of mesenchymal cells was also impaired. As in the axolotl, hedgehog signaling is required for multiple steps in tail regeneration in the Xenopus tadpole, although the location of the Shh source is quite different between the two species. This difference in Shh localization is the likely basis for the differing tissue requirement for tail regeneration between urodeles and anurans.

Notochord-derived hedgehog is essential for tail regeneration in Xenopus tadpole

PubMed Central

2014-01-01

Background Appendage regeneration in amphibians is regulated by the combinatorial actions of signaling molecules. The requirement of molecules secreted from specific tissues is reflected by the observation that the whole process of regeneration can be inhibited if a certain tissue is removed from the amputated stump. Interestingly, urodeles and anurans show different tissue dependencies during tail regeneration. The spinal cord is essential for tail regeneration in urodele but not in anuran larva, whereas the notochord but not the spinal cord is essential for tail regeneration in anuran tadpoles. Sonic hedgehog is one of the signaling molecules responsible for such phenomenon in axolotl, as hedgehog signaling is essential for overall tail regeneration and sonic hedgehog is exclusively expressed in the spinal cord. In order to know whether hedgehog signaling is involved in the molecular mechanism underlying the inconsistent tissue dependency for tail regeneration between anurans and urodeles, we investigated expression of hedgehog signal-related genes in the regenerating tail of Xenopus tadpole and examined the effect of the hedgehog signal inhibitor, cyclopamine, on the tail regeneration. Results In Xenopus, sonic hedgehog is expressed exclusively in the notochord but not in the spinal cord of the regenerate. Overall regeneration was severely impaired in cyclopamine-treated tadpoles. Notochord maturation in the regenerate, including cell alignment and vacuolation, and myofiber formation were inhibited. Proliferation of spinal cord cells in the neural ampulla and of mesenchymal cells was also impaired. Conclusion As in the axolotl, hedgehog signaling is required for multiple steps in tail regeneration in the Xenopus tadpole, although the location of the Shh source is quite different between the two species. This difference in Shh localization is the likely basis for the differing tissue requirement for tail regeneration between urodeles and anurans. PMID:24941877

OCT imaging of craniofacial anatomy in xenopus embryos (Conference Presentation)

NASA Astrophysics Data System (ADS)

Deniz, Engin; Jonas, Stephan M.; Griffin, John; Hooper, Michael C.; Choma, Michael A.; Khokha, Mustafa K.

2016-03-01

The etiology of craniofacial defects is incompletely understood. The ability to obtain large amounts of gene sequence data from families affected by craniofacial defects is opening up new ways to understand molecular genetic etiological factors. One important link between gene sequence data and clinical relevance is biological research into candidate genes and molecular pathways. We present our recent research using OCT as a nondestructive phenotyping modality of craniofacial morphology in Xenopus embryos, an important animal model for biological research in gene and pathway discovery. We define 2D and 3D scanning protocols for a standardized approach to craniofacial imaging in Xenopus embryos. We define standard views and planar reconstructions for visualizing normal anatomy and landmarks. We compare these views and reconstructions to traditional histopathology using alcian blue staining. In addition to being 3D, nondestructive, and having much faster throughout, OCT can identify craniofacial features that are lost during traditional histopathological preparation. We also identify quantitative morphometric parameters to define normative craniofacial anatomy. We also note that craniofacial and cardiac defects are not infrequently present in the same patient (e.g velocardiofacial syndrome). Given that OCT excels at certain aspects of cardiac imaging in Xenopus embryos, our work highlights the potential of using OCT and Xenopus to study molecular genetic factors that impact both cardiac and craniofacial development.

Short toxin-like proteins abound in Cnidaria genomes.

PubMed

Tirosh, Yitshak; Linial, Itai; Askenazi, Manor; Linial, Michal

2012-11-16

Cnidaria is a rich phylum that includes thousands of marine species. In this study, we focused on Anthozoa and Hydrozoa that are represented by the Nematostella vectensis (Sea anemone) and Hydra magnipapillata genomes. We present a method for ranking the toxin-like candidates from complete proteomes of Cnidaria. Toxin-like functions were revealed using ClanTox, a statistical machine-learning predictor trained on ion channel inhibitors from venomous animals. Fundamental features that were emphasized in training ClanTox include cysteines and their spacing along the sequences. Among the 83,000 proteins derived from Cnidaria representatives, we found 170 candidates that fulfill the properties of toxin-like-proteins, the vast majority of which were previously unrecognized as toxins. An additional 394 short proteins exhibit characteristics of toxin-like proteins at a moderate degree of confidence. Remarkably, only 11% of the predicted toxin-like proteins were previously classified as toxins. Based on our prediction methodology and manual annotation, we inferred functions for over 400 of these proteins. Such functions include protease inhibitors, membrane pore formation, ion channel blockers and metal binding proteins. Many of the proteins belong to small families of paralogs. We conclude that the evolutionary expansion of toxin-like proteins in Cnidaria contributes to their fitness in the complex environment of the aquatic ecosystem.

Actin and microtubule-based cytoskeletal cues direct polarized targeting of proteins in neurons

PubMed Central

Arnold, Don B.

2010-01-01

Neuronal proteins are transported to either the axon or dendrites through the action of kinesin motors; however understanding of how cytoskeletal elements steer these cargo-motor complexes to one compartment or the other has remained elusive. Three recent developments, the discovery of an actin-based filter within the axon initial segment, the identification of the pivotal role played by myosin motors in dendritic targeting, and the determination of the properties of a kinesin motor that cause it to prefer axonal to dendritic microtubules, have now provided a structural framework for understanding polarized targeting in neurons. PMID:19671926

Polystyrene nanoparticles affect Xenopus laevis development

NASA Astrophysics Data System (ADS)

Tussellino, Margherita; Ronca, Raffaele; Formiggini, Fabio; Marco, Nadia De; Fusco, Sabato; Netti, Paolo Antonio; Carotenuto, Rosa

2015-02-01

Exposing living organisms to nanoparticulates is potentially hazardous, in particular when it takes place during embryogenesis. In this investigation, we have studied the effects of 50-nm-uncoated polystyrene nanoparticles (PSNPs) as a model to investigate the suitability of their possible future employments. We have used the standardized Frog Embryo Teratogenesis Assay- Xenopus test during the early stages of larval development of Xenopus laevis, and we have employed either contact exposure or microinjections. We found that the embryos mortality rate is dose dependent and that the survived embryos showed high percentage of malformations. They display disorders in pigmentation distribution, malformations of the head, gut and tail, edema in the anterior ventral region, and a shorter body length compared with sibling untreated embryos. Moreover, these embryos grow more slowly than the untreated embryos. Expressions of the mesoderm markers, bra (T-box Brachyury gene), myod1 (myogenic differentiation1), and of neural crest marker sox9 (sex SRY (determining region Y-box 9) transcription factor sox9), are modified. Confocal microscopy showed that the nanoparticles are localized in the cytoplasm, in the nucleus, and in the periphery of the digestive gut cells. Our data suggest that PSNPs are toxic and show a potential teratogenic effect for Xenopus larvae. We hypothesize that these effects may be due either to the amount of NPs that penetrate into the cells and/or to the "corona" effect caused by the interaction of PSNPs with cytoplasm components. The three endpoints of our study, i.e., mortality, malformations, and growth inhibition, suggest that the tests we used may be a powerful and flexible bioassay in evaluating pollutants in aquatic embryos.

Insulin-like growth factor 1 regulation of proliferation and differentiation of Xenopus laevis myogenic cells in vitro.

PubMed

Miyata, Sairi; Yada, Tomotaka; Ishikawa, Natsuko; Taheruzzaman, Kazi; Hara, Ryohei; Matsuzaki, Takashi; Nishikawa, Akio

2017-03-01

To understand the mechanism of muscle remodeling during Xenopus laevis metamorphosis, we examined the in vitro effect of insulin-like growth factor 1 (IGF-1) on growth and differentiation of three different-fate myogenic cell populations: tadpole tail, tadpole dorsal, and young adult leg muscle. IGF-1 promoted growth and differentiation of both tail and leg myogenic cells only under conditions where these cells could proliferate. Inhibition of cell proliferation by DNA synthesis inhibitor cytosine arabinoside completely canceled the IGF-1's cell differentiation promotion, suggesting the possibility that IGF-1's differentiation-promotion effect is an indirect effect via IGF-1's cell proliferation promotion. IGF-1 promoted differentiation dose dependently with maximum effect at 100-500 ng/ml. RT-PCR analysis revealed the upregulation (11-fold) of ifg1 mRNA expression in developing limbs, suggesting that IGF-1 plays a role in promoting muscle differentiation during limb development. The combined effect of triiodo-L-thyronine (T 3 ) and IGF-1 was also examined. In adult leg cells, IGF-1 promoted growth and differentiation irrespective of the presence of T 3 . In larval tail cells, cell count was 76% lower in the presence of T 3 , and IGF-1 did not promote proliferation and differentiation in T 3 -containing medium. In larval dorsal cells, cell count was also lower in the presence of T 3 , but IGF-1 enhanced proliferation and differentiation in T 3 -containing medium. This result is likely due to the presence among dorsal cells of both adult and larval types (1:1). Thus, IGF-1 affects only adult-type myogenic cells in the presence of T 3 and helps accelerate dorsal muscle remodeling during metamorphosis.

Probing the biology of cell boundary conditions through confinement of Xenopus cell-free cytoplasmic extracts.

PubMed

Bermudez, Jessica G; Chen, Hui; Einstein, Lily C; Good, Matthew C

2017-01-01

Cell-free cytoplasmic extracts prepared from Xenopus eggs and embryos have for decades provided a biochemical system with which to interrogate complex cell biological processes in vitro. Recently, the application of microfabrication and microfluidic strategies in biology has narrowed the gap between in vitro and in vivo studies by enabling formation of cell-size compartments containing functional cytoplasm. These approaches provide numerous advantages over traditional biochemical experiments performed in a test tube. Most notably, the cell-free cytoplasm is confined using a two- or three-dimensional boundary, which mimics the natural configuration of a cell. This strategy enables characterization of the spatial organization of a cell, and the role that boundaries play in regulating intracellular assembly and function. In this review, we describe the marriage of Xenopus cell-free cytoplasm and confinement technologies to generate synthetic cell-like systems, the recent biological insights they have enabled, and the promise they hold for future scientific discovery. © 2017 Wiley Periodicals, Inc.

Short Toxin-like Proteins Abound in Cnidaria Genomes

PubMed Central

Tirosh, Yitshak; Linial, Itai; Askenazi, Manor; Linial, Michal

2012-01-01

Cnidaria is a rich phylum that includes thousands of marine species. In this study, we focused on Anthozoa and Hydrozoa that are represented by the Nematostella vectensis (Sea anemone) and Hydra magnipapillata genomes. We present a method for ranking the toxin-like candidates from complete proteomes of Cnidaria. Toxin-like functions were revealed using ClanTox, a statistical machine-learning predictor trained on ion channel inhibitors from venomous animals. Fundamental features that were emphasized in training ClanTox include cysteines and their spacing along the sequences. Among the 83,000 proteins derived from Cnidaria representatives, we found 170 candidates that fulfill the properties of toxin-like-proteins, the vast majority of which were previously unrecognized as toxins. An additional 394 short proteins exhibit characteristics of toxin-like proteins at a moderate degree of confidence. Remarkably, only 11% of the predicted toxin-like proteins were previously classified as toxins. Based on our prediction methodology and manual annotation, we inferred functions for over 400 of these proteins. Such functions include protease inhibitors, membrane pore formation, ion channel blockers and metal binding proteins. Many of the proteins belong to small families of paralogs. We conclude that the evolutionary expansion of toxin-like proteins in Cnidaria contributes to their fitness in the complex environment of the aquatic ecosystem. PMID:23202321

Cloning and functional characterization of the Xenopus orthologue of the Treacher Collins syndrome (TCOF1) gene product.

PubMed

Gonzales, Bianca; Yang, Hushan; Henning, Dale; Valdez, Benigno C

2005-10-10

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development caused by mutations in the TCOF1 gene, which encodes the nucleolar phosphoprotein treacle. We previously reported a function for mammalian treacle in ribosomal DNA gene transcription by its interaction with upstream binding factor. As an initial step in the development of a TCS model for frog the cDNA that encodes the Xenopus laevis treacle was cloned. Although the derived amino acid sequence shows a poor homology with its mammalian orthologues, Xenopus treacle has 11 highly homologous direct repeats near the center of the protein molecule similar to those present in its human, dog and mouse orthologues. Comparison of their amino acid compositions indicates conservation of predominant specific amino acid residues. Antisense-mediated down-regulation of treacle expression in X. laevis oocytes resulted in inhibition of rDNA gene transcription. The results suggest evolutionary conservation of the function of treacle in ribosomal RNA biogenesis in higher eukaryotes.

Assembly of viral particles in Xenopus oocytes: pre-surface-antigens regulate secretion of the hepatitis B viral surface envelope particle.

PubMed Central

Standring, D N; Ou, J H; Rutter, W J

1986-01-01

Infection with hepatitis B virus (HBV) is associated with the production of a viral envelope particle that contains membrane lipids, surface antigen (S), and two presurface-antigens (pre-S) comprised of the entire S moiety with approximately 55 (pre-S2) and 174 (pre-S1) additional NH2-terminal amino acids. We show here that Xenopus oocytes injected with synthetic S mRNA assemble and secrete characteristic 22-nm viral envelope particles. In contrast, pre-S1 and pre-S2 antigens are synthesized but not secreted. By coinjecting mRNAs, we found that synthesis of high levels of pre-S proteins specifically inhibits S antigen secretion. On the other hand, high levels of S synthesis can drive the secretion of small amounts of either pre-S antigen. These observations are consistent with a model for viral envelope assembly in which both S and pre-S proteins are incorporated into a multimeric particle, presumably via interactions between the S protein domains, while the pre-S amino-terminal moieties regulate the secretion of this structure. Our results indicate that Xenopus oocytes will provide a powerful system for studying the morphogenesis of simple structures of viral or cellular origin. Images PMID:3467308

Prions and prion-like proteins.

PubMed

Fraser, Paul E

2014-07-18

Prions are self-replicating protein aggregates and are the primary causative factor in a number of neurological diseases in mammals. The prion protein (PrP) undergoes a conformational transformation leading to aggregation into an infectious cellular pathogen. Prion-like protein spreading and transmission of aggregates between cells have also been demonstrated for other proteins associated with Alzheimer disease and Parkinson disease. This protein-only phenomenon may therefore have broader implications in neurodegenerative disorders. The minireviews in this thematic series highlight the recent advances in prion biology and the roles these unique proteins play in disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Expression of XNOA 36 in the mitochondrial cloud of Xenopus laevis oocytes.

PubMed

Vaccaro, M C; Wilding, M; Dale, B; Campanella, C; Carotenuto, R

2012-08-01

In Xenopus laevis oocytes a mitochondrial cloud (MC) is found between the nucleus and the plasma membrane at stages I-II of oogenesis. The MC contains RNAs that are transported to the future vegetal pole at stage II of oogenesis. In particular, germinal plasm mRNAs are found in the Message Transport Organiser (METRO) region, the MC region opposite to the nucleus. At stages II-III, a second pathway transports Vg1 and VegT mRNAs to the area where the MC content merges with the vegetal cortex. Microtubules become polarized at the sites of migration of Vg1 and VegT mRNAs through an unknown signalling mechanism. In early meiotic stages, the centrioles are almost completely lost with their remnants being dispersed into the cytoplasm and the MC, which may contain a MTOC to be used in the later localization pathway of the mRNAs. In mammals, XNOA 36 encodes a member of a highly conserved protein family and localises to the nucleolus or in the centromeres. In the Xenopus late stage I oocyte, XNOA 36 mRNA is transiently segregated in one half of the oocyte, anchored by a cytoskeletal network that contains spectrin. Here we found that XNOA 36 transcript also localises to the nucleoli and in the METRO region. XNOA 36 protein immunolocalization, using an antibody employed for the library immunoscreening that depicted XNOA 36 expression colonies, labels the migrating MC, the cytoplasm of stage I oocytes and in particular the vegetal cortex facing the MC. The possible role of XNOA 36 in mRNA anchoring to the vegetal cortex or in participating in early microtubule reorganization is discussed.

Purification of Xenopus laevis mitochondrial RNA polymerase and identification of a dissociable factor required for specific transcription

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bogenhagen, D.F.; Insdorf, N.F.

1988-07-01

The Xenopus laevis mitochondrial RNA (mtRNA) polymerase was purified to near homogeneity with an overall yield approaching 50%. The major polypeptides in the final fraction were a doublet of proteins of approximately 140 kilodaltons that copurified with the mtRNA polymerase activity. It appeared likely that the smaller polypeptide is a breakdown product of the larger one. The highly purified polymerase was active in nonspecific transcription but required a dissociable factor for specific transcription of X. laevis mtDNA. The factor could be resolved from mtRNA polymerase by hydrophobic chromatography and had a sedimentation coefficient of 3.0 S. The transcription factor elutedmore » from both the hydrophobic column and a Mono Q anion-exchange column as a single symmetrical peak. The mtRNA polymerase and this factor together are necessary and sufficient for active transcription from four promoters located in a noncoding region of the mtDNA genome between the gene for tRNA/sup Phe/ and the displacement loop.« less

Characteristics of concatemeric GABAA receptors containing α4/δ subunits expressed in Xenopus oocytes

PubMed Central

Shu, Hong-Jin; Bracamontes, John; Taylor, Amanda; Wu, Kyle; Eaton, Megan M; Akk, Gustav; Manion, Brad; Evers, Alex S; Krishnan, Kathiresan; Covey, Douglas F; Zorumski, Charles F; Steinbach, Joe Henry; Mennerick, Steven

2012-01-01

BACKGROUND AND PURPOSE GABAA receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, α4 and δ subunits are key components of extrasynaptic GABAA receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant δ subunits can be difficult to express in heterologous systems. EXPERIMENTAL APPROACH We engineered concatemeric (fused) subunits to ensure δ and α4 subunit expression. We tested the pharmacology of the concatemeric receptors, compared with a common synaptic-like receptor subunit combination (α1 +β2 +γ2L), and with free-subunit α4/δ receptors, expressed in Xenopus oocytes. KEY RESULTS δ-β2 −α4 +β2-α4 cRNA co-injected into Xenopus oocytes resulted in GABA-gated currents with the expected pharmacological properties of α4/δ-containing receptors. Criteria included sensitivity to agonists of different efficacy, sensitivity to the allosteric activator pentobarbital, and modulation of agonist responses by DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide; a δ-selective positive modulator), furosemide, and Zn2+. We used the concatemers to examine neurosteroid sensitivity of extrasynaptic-like, δ-containing receptors. We found no qualitative differences between extrasynaptic-like receptors and synaptic-like receptors in the actions of either negative or positive neurosteroid modulators of receptor function. Quantitative differences were explained by the partial agonist effects of the natural agonist GABA and by a mildly increased sensitivity to low steroid concentrations. CONCLUSIONS AND IMPLICATIONS The neurosteroid structure-activity profile for α4/δ-containing extrasynaptic receptors is unlikely to differ from that of synaptic-like receptors such as α1/β2/γ2-containing receptors. PMID:21950777

The B-subdomain of the Xenopus laevis XFIN KRAB-AB domain is responsible for its weaker transcriptional repressor activity compared to human ZNF10/Kox1.

PubMed

Born, Nadine; Thiesen, Hans-Jürgen; Lorenz, Peter

2014-01-01

The Krüppel-associated box (KRAB) domain interacts with the nuclear hub protein TRIM28 to initiate or mediate chromatin-dependent processes like transcriptional repression, imprinting or suppression of endogenous retroviruses. The prototype KRAB domain initially identified in ZNF10/KOX1 encompasses two subdomains A and B that are found in hundreds of zinc finger transcription factors studied in human and murine genomes. Here we demonstrate for the first time transcriptional repressor activity of an amphibian KRAB domain. After sequence correction, the updated KRAB-AB domain of zinc finger protein XFIN from the frog Xenopus laevis was found to confer transcriptional repression in reporter assays in Xenopus laevis A6 kidney cells as well as in human HeLa, but not in the minnow Pimephales promelas fish cell line EPC. Binding of the XFIN KRAB-AB domain to human TRIM28 was demonstrated in a classical co-immunoprecipitation approach and visualized in a single-cell compartmentalization assay. XFIN-AB displayed reduced potency in repression as well as lower strength of interaction with TRIM28 compared to ZNF10 KRAB-AB. KRAB-B subdomain swapping between the two KRAB domains indicated that it was mainly the KRAB-B subdomain of XFIN that was responsible for its lower capacity in repression and binding to human TRIM28. In EPC fish cells, ZNF10 and XFIN KRAB repressor activity could be partially restored to low levels by adding exogenous human TRIM28. In contrast to XFIN, we did not find any transcriptional repression activity for the KRAB-like domain of human PRDM9 in HeLa cells. PRDM9 is thought to harbor an evolutionary older domain related to KRAB whose homologs even occur in invertebrates. Our results support the notion that functional bona fide KRAB domains which confer transcriptional repression and interact with TRIM28 most likely co-evolved together with TRIM28 at the beginning of tetrapode evolution.

Biochemical study of prolactin binding sites in Xenopus laevis brain and choroid plexus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muccioli, G.; Guardabassi, A.; Pattono, P.

1990-03-01

The occurrence of prolactin binding sites in some brain structures (telencephalon, ventral hypothalamus, myelencephalon, hypophysis, and choroid plexus) from Xenopus laevis (anuran amphibian) was studied by the in vitro biochemical technique. The higher binding values were obtained at the level of the choroid plexus and above all of the hypothalamus. On the bases of hormonal specificity and high affinity, these binding sites are very similar to those of prolactin receptors of classical target tissues as well as of those described by us in other structures from Xenopus. To our knowledge, the present results provide the first demonstration of the occurrencemore » of prolactin specific binding sites in Xenopus laevis choroid plexus cells.« less

Expression profiles of the Gα subunits during Xenopus tropicalis embryonic development.

PubMed

Fuentealba, Jaime; Toro-Tapia, Gabriela; Rodriguez, Marion; Arriagada, Cecilia; Maureira, Alejandro; Beyer, Andrea; Villaseca, Soraya; Leal, Juan I; Hinrichs, Maria V; Olate, Juan; Caprile, Teresa; Torrejón, Marcela

2016-09-01

Heterotrimeric G protein signaling plays major roles during different cellular events. However, there is a limited understanding of the molecular mechanisms underlying G protein control during embryogenesis. G proteins are highly conserved and can be grouped into four subfamilies according to sequence homology and function. To further studies on G protein function during embryogenesis, the present analysis identified four Gα subunits representative of the different subfamilies and determined their spatiotemporal expression patterns during Xenopus tropicalis embryogenesis. Each of the Gα subunit transcripts was maternally and zygotically expressed, and, as development progressed, dynamic expression patterns were observed. In the early developmental stages, the Gα subunits were expressed in the animal hemisphere and dorsal marginal zone. While expression was observed at the somite boundaries, in vascular structures, in the eye, and in the otic vesicle during the later stages, expression was mainly found in neural tissues, such as the neural tube and, especially, in the cephalic vesicles, neural crest region, and neural crest-derived structures. Together, these results support the pleiotropism and complexity of G protein subfamily functions in different cellular events. The present study constitutes the most comprehensive description to date of the spatiotemporal expression patterns of Gα subunits during vertebrate development. Copyright © 2016 Elsevier B.V. All rights reserved.

Velocity and Drag Forces on motor-protein-driven Vesicles in Cells

NASA Astrophysics Data System (ADS)

Hill, David; Holzwarth, George; Bonin, Keith

2002-10-01

In cells, vesicle transport is driven by motor proteins such as kinesin and dynein, which use the chemical energy of ATP to overcome drag. Using video-enhanced DIC microscopy at 8 frames/s, we find that vesicles in PC12 neurites move with an average velocity of 1.52 0.66 μm/s. The drag force and work required for such steady movement, calculated from Stokes' Law and the zero-frequency viscosity of the cytoplasm, suggest that multiple motors are required to move one vesicle. In buffer, single kinesin molecules move beads in 8-nm steps, each step taking only 50 μs [1]. The effects of such quick steps in cytoplasm, using viscoelastic moduli of COS7 cells, are small [2]. To measure drag forces more directly, we are using B-field-driven magnetic beads in PC12 cells to mimic kinesin-driven vesicles. [1] Nishiyama, M. et al., Nat. Cell Bio. 3, 425-428 (2001). [2] Holzwarth, Bonin, and Hill, Biophys J 82, 1784-1790 (2002).

XENOPUS LAEVIS: A CULTURING AND REARING PROTOCOL

EPA Science Inventory

Xenopus laevis are used extensively here at MED-Duluth as a model for assessing development toxicity to xenobiotics. As a result, a culturing system has been developed that provides eggs and tadpoles of consistent high quality for use by researchers at the facility. The methods ...

Xenopus laevis in Developmental and Molecular Biology.

ERIC Educational Resources Information Center

Dawid, Igor B.; Sargent, Thomas D.

1988-01-01

Discusses the advantages of Xenopus laevis as an experimental animal in the study of embryogenesis in vertebrates. Summarizes the contributions of this system to the analysis of ribosomal and 5S RNA genes, and the diverse and highly productive applications of the oocyte injection technology. (RT)

Inositol-requiring enzyme 1α is required for gut development in Xenopus lavies embryos

PubMed Central

Guo, Jing; Li, Xin-Xin; Feng, Jiao-Jiao; Yin, Chen-Yang; Wang, Xue-Jun; Wang, Ning; Yuan, Li

2013-01-01

AIM: To investigate the role of inositol-requiring enzyme 1α (IRE1α) in gut development of Xenopus lavies embryos. METHODS: Xenopus embryos were obtained with in vitro fertilization and cultured in 0.1 × MBSH. One and half nanogram of IRE1α, 1 ng of IRE1α-GR mRNA, 1 ng of IRE1αΔC-GR mRNA, and 50 ng of IRE1α morpholino oligonucleotide (MO) or XBP1(C)MO were injected into four blastomeres at 4-cell stage for scoring the phenotype and marker gene analysis. To rescue the effect of IRE1α MO, 1 ng of IRE1α-GR mRNA was co-injected with 50 ng of MO. For the activation of the GR-fusion proteins, dexamethasone was prepared as 5 mmol/L stock solutions in 100% ethanol and applied to the mRNA injected embryos at desired stages in a concentration of 10 μmol/L in 0.1 × MBSH. Embryos were kept in dexamethasone up to stage 41. Whole-mount in situ hybridization was used to determine specific gene expression, such as IRE1α, IRE1β, Xbra and Xsox17α. IRE1α protein expression during Xenopus embryogenesis was detected by Western blotting. RESULTS: In the whole-mount in situ hybridization analysis, xenopus IRE1α and IRE1β showed quite different expression pattern during tadpole stage. The relatively higher expression of IRE1α was observed in the pancreas, and significant transcription of IRE1β was found in the liver. IRE1α protein could be detected at all developmental stages analyzed, from stage 1 to stage 42. Gain-of-function assay showed that IRE1α mRNA injected embryos at tailbud stage were nearly normal and the expression of the pan-mesodermal marker gene Xbra and the endodermal gene Xsox17α at stage 10.5 was not significantly changed in embryos injected with IRE1α mRNA as compared to uninjected control embryos. And at tadpole stage, the embryos injected with IRE1α-GR mRNA did not display overt phenotype, such as gut-coiling defect. Loss-of-function assay demonstrated that the IRE1α MO injected embryos were morphologically normal before the tailbud stages

Inositol-requiring enzyme 1α is required for gut development in Xenopus lavies embryos.

PubMed

Guo, Jing; Li, Xin-Xin; Feng, Jiao-Jiao; Yin, Chen-Yang; Wang, Xue-Jun; Wang, Ning; Yuan, Li

2013-01-14

To investigate the role of inositol-requiring enzyme 1α (IRE1α) in gut development of Xenopus lavies embryos. Xenopus embryos were obtained with in vitro fertilization and cultured in 0.1 × MBSH. One and half nanogram of IRE1α, 1 ng of IRE1α-GR mRNA, 1 ng of IRE1αΔC-GR mRNA, and 50 ng of IRE1α morpholino oligonucleotide (MO) or XBP1(C)MO were injected into four blastomeres at 4-cell stage for scoring the phenotype and marker gene analysis. To rescue the effect of IRE1α MO, 1 ng of IRE1α-GR mRNA was co-injected with 50 ng of MO. For the activation of the GR-fusion proteins, dexamethasone was prepared as 5 mmol/L stock solutions in 100% ethanol and applied to the mRNA injected embryos at desired stages in a concentration of 10 μmol/L in 0.1 × MBSH. Embryos were kept in dexamethasone up to stage 41. Whole-mount in situ hybridization was used to determine specific gene expression, such as IRE1α, IRE1β, Xbra and Xsox17α. IRE1α protein expression during Xenopus embryogenesis was detected by Western blotting. In the whole-mount in situ hybridization analysis, xenopus IRE1α and IRE1β showed quite different expression pattern during tadpole stage. The relatively higher expression of IRE1α was observed in the pancreas, and significant transcription of IRE1β was found in the liver. IRE1α protein could be detected at all developmental stages analyzed, from stage 1 to stage 42. Gain-of-function assay showed that IRE1α mRNA injected embryos at tailbud stage were nearly normal and the expression of the pan-mesodermal marker gene Xbra and the endodermal gene Xsox17α at stage 10.5 was not significantly changed in embryos injected with IRE1α mRNA as compared to uninjected control embryos. And at tadpole stage, the embryos injected with IRE1α-GR mRNA did not display overt phenotype, such as gut-coiling defect. Loss-of-function assay demonstrated that the IRE1α MO injected embryos were morphologically normal before the tailbud stages. We did not observe a

Involvement of the kinesin family members KIF4A and KIF5C in intellectual disability and synaptic function.

PubMed

Willemsen, Marjolein H; Ba, Wei; Wissink-Lindhout, Willemijn M; de Brouwer, Arjan P M; Haas, Stefan A; Bienek, Melanie; Hu, Hao; Vissers, Lisenka E L M; van Bokhoven, Hans; Kalscheuer, Vera; Nadif Kasri, Nael; Kleefstra, Tjitske

2014-07-01

Kinesin superfamily (KIF) genes encode motor proteins that have fundamental roles in brain functioning, development, survival and plasticity by regulating the transport of cargo along microtubules within axons, dendrites and synapses. Mouse knockout studies support these important functions in the nervous system. The role of KIF genes in intellectual disability (ID) has so far received limited attention, although previous studies have suggested that many ID genes impinge on synaptic function. By applying next-generation sequencing (NGS) in ID patients, we identified likely pathogenic mutations in KIF4A and KIF5C. To further confirm the pathogenicity of these mutations, we performed functional studies at the level of synaptic function in primary rat hippocampal neurons. Four males from a single family with a disruptive mutation in the X-linked KIF4A (c.1489-8_1490delins10; p.?- exon skipping) showed mild to moderate ID and epilepsy. A female patient with a de novo missense mutation in KIF5C (c.11465A>C; p.(Glu237Lys)) presented with severe ID, epilepsy, microcephaly and cortical malformation. Knock-down of Kif4a in rat primary hippocampal neurons altered the balance between excitatory and inhibitory synaptic transmission, whereas the mutation in Kif5c affected its protein function at excitatory synapses. Our results suggest that mutations in KIF4A and KIF5C cause ID by tipping the balance between excitatory and inhibitory synaptic excitability. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Accelerated Gene Evolution and Subfunctionalization in thePseudotetraploid Frog Xenopus Laevis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hellsten, Uffe; Khokha, Mustafa K.; Grammar, Timothy C.

2007-03-01

Ancient whole genome duplications have been implicated in the vertebrate and teleost radiations, and in the emergence of diverse angiosperm lineages, but the evolutionary response to such a perturbation is still poorly understood. The African clawed frog Xenopus laevis experienced a relatively recent tetraploidization {approx} 40 million years ago. Analysis of the considerable amount of EST sequence available for this species together with the genome sequence of the related diploid Xenopus tropicalis provides a unique opportunity to study the genomic response to whole genome duplication.

Anxa4 Genes are Expressed in Distinct Organ Systems in Xenopus laevis and tropicalis But are Functionally Conserved

PubMed Central

Massé, Karine L; Collins, Robert J; Bhamra, Surinder; Seville, Rachel A

2007-01-01

Anxa4 belongs to the multigenic annexin family of proteins which are characterized by their ability to interact with membranes in a calcium-dependent manner. Defined as a marker for polarized epithelial cells, Anxa4 is believed to be involved in many cellular processes but its functions in vivo are still poorly understood. Previously, we cloned Xanx4 in Xenopus laevis (now referred to as anxa4a) and demonstrated its role during organogenesis of the pronephros, providing the first evidence of a specific function for this protein during the development of a vertebrate. Here, we describe the strict conservation of protein sequence and functional domains of anxa4 during vertebrate evolution. We also identify the paralog of anxa4a, anxa4b and show its specific temporal and spatial expression pattern is different from anxa4a. We show that anxa4 orthologs in X. laevis and tropicalis display expression domains in different organ systems. Whilst the anxa4a gene is mainly expressed in the kidney, Xt anxa4 is expressed in the liver. Finally, we demonstrate Xt anxa4 and anxa4a can display conserved function during kidney organogenesis, despite the fact that Xt anxa4 transcripts are not expressed in this domain. This study highlights the divergence of expression of homologous genes during Xenopus evolution and raises the potential problems of using X. tropicalis promoters in X. laevis. PMID:19279706

In vivo Assessment and Potential Diagnosis of Xenobiotics that Perturb the Thyroid Pathway: Proteomic Analysis of Xenopus laevis Brain Tissue following Exposure to Model T4 Inhibitors

EPA Science Inventory

As part of a multi-endpoint systems approach to develop comprehensive methods for assessing endocrine stressors in vertebrates, differential protein profiling was used to investigate expression profiles in the brain of an amphibian model (Xenopus laevis) following in vivo exposur...

Sperm and Spermatids Contain Different Proteins and Bind Distinct Egg Factors

PubMed Central

Teperek, Marta; Miyamoto, Kei; Simeone, Angela; Feret, Renata; Deery, Michael J.; Gurdon, John B.; Jullien, Jerome

2014-01-01

Spermatozoa are more efficient at supporting normal embryonic development than spermatids, their immature, immediate precursors. This suggests that the sperm acquires the ability to support embryonic development during spermiogenesis (spermatid to sperm maturation). Here, using Xenopus laevis as a model organism, we performed 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry analysis of differentially expressed proteins between sperm and spermatids in order to identify factors that could be responsible for the efficiency of the sperm to support embryonic development. Furthermore, benefiting from the availability of egg extracts in Xenopus, we also tested whether the chromatin of sperm could attract different egg factors compared to the chromatin of spermatids. Our analysis identified: (1) several proteins which were present exclusively in sperm; but not in spermatid nuclei and (2) numerous egg proteins binding to the sperm (but not to the spermatid chromatin) after incubation in egg extracts. Amongst these factors we identified many chromatin-associated proteins and transcriptional repressors. Presence of transcriptional repressors binding specifically to sperm chromatin could suggest its preparation for the early embryonic cell cycles, during which no transcription is observed and suggests that sperm chromatin has a unique protein composition, which facilitates the recruitment of egg chromatin remodelling factors. It is therefore likely that the acquisition of these sperm-specific factors during spermiogenesis makes the sperm chromatin suitable to interact with the maternal factors and, as a consequence, to support efficient embryonic development. PMID:25244019

Activin-like signal activates dorsal-specific maternal RNA between 8- and 16-cell stages of Xenopus.

PubMed

Hainski, A M; Moody, S A

1996-01-01

In many animals the dorsal-ventral axis forms by an initial localization of maternal molecules, which then regulate the spatial location of signals that directly influence the expression of axis-specific fates. Several recent studies have demonstrated that dorsal-animal blastomeres of the Xenopus morula (8-32 cells) are biased toward dorsal fates prior to mesoderm inductive signaling. In this study we ask whether the dorsal bias is the result of autonomous expression of maternal molecules specifically localized within dorsal cells or of early activating signals. It was found that although 16-cell dorsal-animal blastomeres (D1.1) can differentiate into dorsal tissues when cultured alone, the 8-cell mothers (D1) can not. Likewise, although RNA extracted from D1.1 can induce an extra dorsal axis when injected into vegetal blastomeres, RNA extracted from D1 can not. However, D1 does express dorsal tissues if co-cultured with dorsal-vegetal cells or with culture medium containing a mixture of activins (PIF-medium). Furthermore, short-term culture of D1 in PIF-medium enables the D1 RNA to induce an ectopic dorsal axis. Ventral-animal blastomeres also can express dorsal axial tissues when co-cultured with dorsal-vegetal blastomeres or in PIF-medium, but the RNA from the activin-treated ventral cells cannot induce ectopic dorsal axes. These studies demonstrate that there are maternal RNAs that, shortly after fertilization, are present only in the dorsal-animal region. They do not act cell autonomously, but require an activin-like signal. These RNAs may function by increasing the responsiveness of dorsal-animal blastomeres to the mesoderm inductive signals present in both the morula and the blastula.

Concentration-dependent Effects of Nuclear Lamins on Nuclear Size in Xenopus and Mammalian Cells*

PubMed Central

Jevtić, Predrag; Edens, Lisa J.; Li, Xiaoyang; Nguyen, Thang; Chen, Pan; Levy, Daniel L.

2015-01-01

A fundamental question in cell biology concerns the regulation of organelle size. While nuclear size is exquisitely controlled in different cell types, inappropriate nuclear enlargement is used to diagnose and stage cancer. Clarifying the functional significance of nuclear size necessitates an understanding of the mechanisms and proteins that control nuclear size. One structural component implicated in the regulation of nuclear morphology is the nuclear lamina, a meshwork of intermediate lamin filaments that lines the inner nuclear membrane. However, there has not been a systematic investigation of how the level and type of lamin expression influences nuclear size, in part due to difficulties in precisely controlling lamin expression levels in vivo. In this study, we circumvent this limitation by studying nuclei in Xenopus laevis egg and embryo extracts, open biochemical systems that allow for precise manipulation of lamin levels by the addition of recombinant proteins. We find that nuclear growth and size are sensitive to the levels of nuclear lamins, with low and high concentrations increasing and decreasing nuclear size, respectively. Interestingly, each type of lamin that we tested (lamins B1, B2, B3, and A) similarly affected nuclear size whether added alone or in combination, suggesting that total lamin concentration, and not lamin type, is more critical to determining nuclear size. Furthermore, we show that altering lamin levels in vivo, both in Xenopus embryos and mammalian tissue culture cells, also impacts nuclear size. These results have implications for normal development and carcinogenesis where both nuclear size and lamin expression levels change. PMID:26429910

Sequencing and analysis of 10,967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis reveals post-tetraploidization transcriptome remodeling

PubMed Central

Morin, Ryan D.; Chang, Elbert; Petrescu, Anca; Liao, Nancy; Griffith, Malachi; Kirkpatrick, Robert; Butterfield, Yaron S.; Young, Alice C.; Stott, Jeffrey; Barber, Sarah; Babakaiff, Ryan; Dickson, Mark C.; Matsuo, Corey; Wong, David; Yang, George S.; Smailus, Duane E.; Wetherby, Keith D.; Kwong, Peggy N.; Grimwood, Jane; Brinkley, Charles P.; Brown-John, Mabel; Reddix-Dugue, Natalie D.; Mayo, Michael; Schmutz, Jeremy; Beland, Jaclyn; Park, Morgan; Gibson, Susan; Olson, Teika; Bouffard, Gerard G.; Tsai, Miranda; Featherstone, Ruth; Chand, Steve; Siddiqui, Asim S.; Jang, Wonhee; Lee, Ed; Klein, Steven L.; Blakesley, Robert W.; Zeeberg, Barry R.; Narasimhan, Sudarshan; Weinstein, John N.; Pennacchio, Christa Prange; Myers, Richard M.; Green, Eric D.; Wagner, Lukas; Gerhard, Daniela S.; Marra, Marco A.; Jones, Steven J.M.; Holt, Robert A.

2006-01-01

Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection Initiative. Here we present 10,967 full ORF verified cDNA clones (8049 from X. laevis and 2918 from X. tropicalis) as a community resource. Because the genome of X. laevis, but not X. tropicalis, has undergone allotetraploidization, comparison of coding sequences from these two clawed (pipid) frogs provides a unique angle for exploring the molecular evolution of duplicate genes. Within our clone set, we have identified 445 gene trios, each comprised of an allotetraploidization-derived X. laevis gene pair and their shared X. tropicalis ortholog. Pairwise dN/dS, comparisons within trios show strong evidence for purifying selection acting on all three members. However, dN/dS ratios between X. laevis gene pairs are elevated relative to their X. tropicalis ortholog. This difference is highly significant and indicates an overall relaxation of selective pressures on duplicated gene pairs. We have found that the paralogs that have been lost since the tetraploidization event are enriched for several molecular functions, but have found no such enrichment in the extant paralogs. Approximately 14% of the paralogous pairs analyzed here also show differential expression indicative of subfunctionalization. PMID:16672307

Expression, Purification, and Structural Insights for the Human Uric Acid Transporter, GLUT9, Using the Xenopus laevis Oocytes System

PubMed Central

Clémençon, Benjamin; Lüscher, Benjamin P.; Fine, Michael; Baumann, Marc U.; Surbek, Daniel V.; Bonny, Olivier; Hediger, Matthias A.

2014-01-01

The urate transporter, GLUT9, is responsible for the basolateral transport of urate in the proximal tubule of human kidneys and in the placenta, playing a central role in uric acid homeostasis. GLUT9 shares the least homology with other members of the glucose transporter family, especially with the glucose transporting members GLUT1-4 and is the only member of the GLUT family to transport urate. The recently published high-resolution structure of XylE, a bacterial D-xylose transporting homologue, yields new insights into the structural foundation of this GLUT family of proteins. While this represents a huge milestone, it is unclear if human GLUT9 can benefit from this advancement through subsequent structural based targeting and mutagenesis. Little progress has been made toward understanding the mechanism of GLUT9 since its discovery in 2000. Before work can begin on resolving the mechanisms of urate transport we must determine methods to express, purify and analyze hGLUT9 using a model system adept in expressing human membrane proteins. Here, we describe the surface expression, purification and isolation of monomeric protein, and functional analysis of recombinant hGLUT9 using the Xenopus laevis oocyte system. In addition, we generated a new homology-based high-resolution model of hGLUT9 from the XylE crystal structure and utilized our purified protein to generate a low-resolution single particle reconstruction. Interestingly, we demonstrate that the functional protein extracted from the Xenopus system fits well with the homology-based model allowing us to generate the predicted urate-binding pocket and pave a path for subsequent mutagenesis and structure-function studies. PMID:25286413

Alzheimer Aβ disrupts the mitotic spindle and directly inhibits mitotic microtubule motors

PubMed Central

Borysov, Sergiy I; Granic, Antoneta; Padmanabhan, Jaya; Walczak, Claire E

2011-01-01

Chromosome mis-segregation and aneuploidy are greatly induced in Alzheimer disease and models thereof by mutant forms of the APP and PS proteins and by their product, the Aβ peptide. Here we employ human somatic cells and Xenopus egg extracts to show that Aβ impairs the assembly and maintenance of the mitotic spindle. Mechanistically, these defects result from Aβ's inhibition of mitotic motor kinesins, including Eg5, KIF4A and MCAK. In vitro studies show that oligomeric Aβ directly inhibits recombinant MCAK by a noncompetitive mechanism. In contrast, inhibition of Eg5 and KIF4A is competitive with respect to both ATP and microtubules, indicating that Aβ interferes with their interactions with the microtubules of the mitotic spindle. Consistently, increased levels of polymerized microtubules or of the microtubule stabilizing protein Tau significantly decrease the inhibitory effect of Aβ on Eg5 and KIF4A. Together, these results indicate that by disrupting the interaction between specific kinesins and microtubules and by exerting a direct inhibitory effect on the motor activity, excess Aβ deregulates the mechanical forces that govern the spindle and thereby leads to the generation of defective mitotic structures. The resulting defect in neurogenesis can account for the over 30% aneuploid/hyperploid, degeneration-prone neurons observed in Alzheimer disease brain. The finding of mitotic motors including Eg5 in mature post-mitotic neurons implies that their inhibition by Aβ may also disrupt neuronal function and plasticity. PMID:21566458

Modulation of K+ currents in Xenopus spinal neurons by p2y receptors: a role for ATP and ADP in motor pattern generation

PubMed Central

Brown, Paul; Dale, Nicholas

2002-01-01

We have investigated the pharmacological properties and targets of p2y purinoceptors in Xenopus embryo spinal neurons. ATP reversibly inhibited the voltage-gated K+ currents by 10 ± 3 %. UTP and the analogues α,β-methylene-ATP and 2-methylthio-ATP also inhibited K+ currents. This agonist profile is similar to that reported for a p2y receptor cloned from Xenopus embryos. Voltage-gated K+ currents could be inhibited by ADP (9 ± 0.8 %) suggesting that a further p2y1-like receptor is also present in the embryo spinal cord. Unexpectedly we found that α,β-methylene-ADP, often used to block the ecto-5′-nucleotidase, also inhibited voltage-gated K+ currents (7 ± 2.3 %). This inhibition was occluded by ADP, suggesting that α,β-methylene-ADP is an agonist at p2y1 receptors. We have directly studied the properties of the ecto-5′-nucleotidase in Xenopus embryo spinal cord. Although ADP inhibited this enzyme, α,β-methylene-ADP had no action. Caution therefore needs to be used when interpreting the actions of α,β-methylene-ADP as it has previously unreported agonist activity at P2 receptors. Xenopus spinal neurons possess fast and slow voltage-gated K+ currents. By using catechol to selectively block the fast current, we completely occluded the actions of ATP and ADP. Furthermore, the purines appeared to block only the fast relaxation component of the tail currents. We therefore conclude that the p2y receptors target only the fast component of the delayed rectifier. As ATP breakdown to ADP is rapid and ADP may accumulate at higher levels than ATP, the contribution of ADP acting through p2y1-like receptors may be an important additional mechanism for the control of spinal motor pattern generation. PMID:11986373

Biochemical effects of chlorpyrifos on two developmental stages of Xenopus laevis.

PubMed

Richards, Sean M; Kendall, Ron J

2002-09-01

Abstract-The effects of a 96-h static exposure to chlorpyrifos were examined in two developmental stages of larval Xenopus laevis (premetamorph and metamorph). Measures of effect included mortality, deformity, cholinesterase (ChE) activity, and DNA and protein concentration. All parameters indicated that metamorphs were more sensitive than were premetamorphs. For larvae exposed as premetamorphs, the median lethal concentration and median effective concentration were 14.6 mg/L and 1.71 mg/L; for those exposed as metamorphs, values were 0.56 mg/L and 0.24 mg/L, respectively. Cholinesterase activity was the most sensitive biochemical parameter. Exposure to chlorpyrifos at 0.01 mg/L caused significant decreases in the ChE activity of metamorphs; 0.1 mg/L significantly decreased premetamorph ChE activity. Metamorph DNA was significantly decreased at 0.1 mg/L; premetamorph DNA was not reduced until exposure to 1.0 mg/L. Whole-body protein was the least sensitive biochemical measure of effect. Premetamorphs did not experience a reduction in protein concentrations. Metamorph protein concentration was significantly decreased at 1.0 mg/L. Based on current surface water data, the most sensitive effect would not have a high probability (< or = 4.2%) of occurring in the environment.

Klp10A modulates the localization of centriole-associated proteins during Drosophila male gametogenesis.

PubMed

Gottardo, Marco; Callaini, Giuliano; Riparbelli, Maria Giovanna

2016-12-16

Mutations in Klp10A, a microtubule-depolymerising Kinesin-13, lead to overly long centrioles in Drosophila male germ cells. We demonstrated that the loss of Klp10A modifies the distribution of typical proteins involved in centriole assembly and function. In the absence of Klp10A the distribution of Drosophila pericentrin-like protein (Dplp), Sas-4 and Sak/Plk4 that are restricted in control testes to the proximal end of the centriole increase along the centriole length. Remarkably, the cartwheel is lacking or it appears abnormal in mutant centrioles, suggesting that this structure may spatially delimit protein localization. Moreover, the parent centrioles that in control cells have the same dimensions grow at different rates in mutant testes with the mother centrioles longer than the daughters. Daughter centrioles have often an ectopic position with respect to the proximal end of the mothers and failed to recruit Dplp.

The Sperm-surface glycoprotein, SGP, is necessary for fertilization in the frog, Xenopus laevis.

PubMed

Nagai, Keita; Ishida, Takuya; Hashimoto, Takafumi; Harada, Yuichirou; Ueno, Shuichi; Ueda, Yasushi; Kubo, Hideo; Iwao, Yasuhiro

2009-06-01

To identify a molecule involved in sperm-egg plasma membrane binding at fertilization, a monoclonal antibody against a sperm-surface glycoprotein (SGP) was obtained by immunizing mice with a sperm membrane fraction of the frog, Xenopus laevis, followed by screening of the culture supernatants based on their inhibitory activity against fertilization. The fertilization of both jellied and denuded eggs was effectively inhibited by pretreatment of sperm with intact anti-SGP antibody as well as its Fab fragment, indicating that the antibody recognizes a molecule on the sperm's surface that is necessary for fertilization. On Western blots, the anti-SGP antibody recognized large molecules, with molecular masses of 65-150 kDa and minor smaller molecules with masses of 20-28 kDa in the sperm membrane vesicles. SGP was distributed over nearly the entire surface of the sperm, probably as an integral membrane protein in close association with microfilaments. More membrane vesicles containing SGP bound to the surface were found in the animal hemisphere compared with the vegetal hemisphere in unfertilized eggs, but the vesicle-binding was not observed in fertilized eggs. These results indicate that SGP mediates sperm-egg membrane binding and is responsible for the establishment of fertilization in Xenopus.

Xenopus egg extract: A powerful tool to study genome maintenance mechanisms.

PubMed

Hoogenboom, Wouter S; Klein Douwel, Daisy; Knipscheer, Puck

2017-08-15

DNA repair pathways are crucial to maintain the integrity of our genome and prevent genetic diseases such as cancer. There are many different types of DNA damage and specific DNA repair mechanisms have evolved to deal with these lesions. In addition to these repair pathways there is an extensive signaling network that regulates processes important for repair, such as cell cycle control and transcription. Despite extensive research, DNA damage repair and signaling are not fully understood. In vitro systems such as the Xenopus egg extract system, have played, and still play, an important role in deciphering the molecular details of these processes. Xenopus laevis egg extracts contain all factors required to efficiently perform DNA repair outside a cell, using mechanisms conserved in humans. These extracts have been used to study several genome maintenance pathways, including mismatch repair, non-homologous end joining, ICL repair, DNA damage checkpoint activation, and replication fork stability. Here we describe how the Xenopus egg extract system, in combination with specifically designed DNA templates, contributed to our detailed understanding of these pathways. Copyright © 2017. Published by Elsevier Inc.

A Crayfish Insulin-like-binding Protein

PubMed Central

Rosen, Ohad; Weil, Simy; Manor, Rivka; Roth, Ziv; Khalaila, Isam; Sagi, Amir

2013-01-01

Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatics analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which according to three-dimensional modeling is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a “pulldown” methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23–38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development. PMID:23775079

A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones.

PubMed

Siebert, Matthias; Böhme, Mathias A; Driller, Jan H; Babikir, Husam; Mampell, Malou M; Rey, Ulises; Ramesh, Niraja; Matkovic, Tanja; Holton, Nicole; Reddy-Alla, Suneel; Göttfert, Fabian; Kamin, Dirk; Quentin, Christine; Klinedinst, Susan; Andlauer, Till Fm; Hell, Stefan W; Collins, Catherine A; Wahl, Markus C; Loll, Bernhard; Sigrist, Stephan J

2015-08-14

Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). We here demonstrate axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. We show in atomic detail that RBP C-terminal SH3 domains bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Pointmutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes.

Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ostrup, Olga, E-mail: osvarcova@gmail.com; Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo; Norwegian Center for Stem Cell Research, Oslo

Highlights: {yields} Xenopus egg extract remodels nuclei and alter cell growth characteristics. {yields} Ribosomal genes are reprogrammed within 6 h after extract exposure. {yields} rDNA reprogramming involves promoter targeting of SNF2H remodeling complex. {yields} Xenopus egg extract does not initiate stress-related response in somatic cells. {yields} Aza-cytidine elicits a stress-induced response in reprogrammed cells. -- Abstract: Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression.more » This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus egg extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.« less

Tissue-Specific Upregulation of MDS/EVI Gene Transcripts in the Intestine by Thyroid Hormone during Xenopus Metamorphosis

PubMed Central

Hasebe, Takashi; Fu, Liezhen; Heimeier, Rachel A.; Das, Biswajit; Ishizuya-Oka, Atsuko; Shi, Yun-Bo

2013-01-01

Background Intestinal remodeling during amphibian metamorphosis resembles the maturation of the adult intestine during mammalian postembryonic development when the adult epithelial self-renewing system is established under the influence of high concentrations of plasma thyroid hormone (T3). This process involves de novo formation and subsequent proliferation and differentiation of the adult stem cells. Methodology/Principal Findings The T3-dependence of the formation of adult intestinal stem cell during Xenopus laevis metamorphosis offers a unique opportunity to identify genes likely important for adult organ-specific stem cell development. We have cloned and characterized the ectopic viral integration site 1 (EVI) and its variant myelodysplastic syndrome 1 (MDS)/EVI generated via transcription from the upstream MDS promoter and alternative splicing. EVI and MDS/EVI have been implicated in a number of cancers including breast, leukemia, ovarian, and intestinal cancers. We show that EVI and MDS/EVI transcripts are upregulated by T3 in the epithelium but not the rest of the intestine in Xenopus laevis when adult stem cells are forming in the epithelium. Conclusions/Significance Our results suggest that EVI and MDS/EVI are likely involved in the development and/or proliferation of newly forming adult intestinal epithelial cells. PMID:23383234

Xenbase, the Xenopus model organism database; new virtualized system, data types and genomes

PubMed Central

Karpinka, J. Brad; Fortriede, Joshua D.; Burns, Kevin A.; James-Zorn, Christina; Ponferrada, Virgilio G.; Lee, Jacqueline; Karimi, Kamran; Zorn, Aaron M.; Vize, Peter D.

2015-01-01

Xenbase (http://www.xenbase.org), the Xenopus frog model organism database, integrates a wide variety of data from this biomedical model genus. Two closely related species are represented: the allotetraploid Xenopus laevis that is widely used for microinjection and tissue explant-based protocols, and the diploid Xenopus tropicalis which is used for genetics and gene targeting. The two species are extremely similar and protocols, reagents and results from each species are often interchangeable. Xenbase imports, indexes, curates and manages data from both species; all of which are mapped via unique IDs and can be queried in either a species-specific or species agnostic manner. All our services have now migrated to a private cloud to achieve better performance and reliability. We have added new content, including providing full support for morpholino reagents, used to inhibit mRNA translation or splicing and binding to regulatory microRNAs. New genomes assembled by the JGI for both species and are displayed in Gbrowse and are also available for searches using BLAST. Researchers can easily navigate from genome content to gene page reports, literature, experimental reagents and many other features using hyperlinks. Xenbase has also greatly expanded image content for figures published in papers describing Xenopus research via PubMedCentral. PMID:25313157

Pattern formation in early embryogenesis of Xenopus laevis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mglinets, V.A.

1995-07-01

Establishment of egg polarity, separation of germ layers, and the appearance of animal-vegetal, dorsoventral, and anteroposterior axes in Xenopus laevis embryos are considered. The control of these processes by gene coding for growth factors, protooncogens, and homeobox-containing genes is also been reviewed.

Calcium Signaling and Meiotic Exit at Fertilization in Xenopus Egg

PubMed Central

Tokmakov, Alexander A.; Stefanov, Vasily E.; Iwasaki, Tetsushi; Sato, Ken-Ichi; Fukami, Yasuo

2014-01-01

Calcium is a universal messenger that mediates egg activation at fertilization in all sexually reproducing species studied. However, signaling pathways leading to calcium generation and the mechanisms of calcium-induced exit from meiotic arrest vary substantially among species. Here, we review the pathways of calcium signaling and the mechanisms of meiotic exit at fertilization in the eggs of the established developmental model, African clawed frog, Xenopus laevis. We also discuss calcium involvement in the early fertilization-induced events in Xenopus egg, such as membrane depolarization, the increase in intracellular pH, cortical granule exocytosis, cortical contraction, contraction wave, cortical rotation, reformation of the nuclear envelope, sperm chromatin decondensation and sister chromatid segregation. PMID:25322156

Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts.

PubMed

Strausfeld, U P; Howell, M; Descombes, P; Chevalier, S; Rempel, R E; Adamczewski, J; Maller, J L; Hunt, T; Blow, J J

1996-06-01

Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define 'S-phase promoting factor' (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclusively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cd2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.

Expression and functional analyses of a Kinesin gene GhKIS13A1 from cotton (Gossypium hirsutum) fiber.

PubMed

Li, Yan-Jun; Zhu, Shou-Hong; Zhang, Xin-Yu; Liu, Yong-Chang; Xue, Fei; Zhao, Lan-Jie; Sun, Jie

2017-06-12

Cotton fiber, a natural fiber widely used in the textile industry, is differentiated from single cell of ovule epidermis. A large number of genes are believed to be involved in fiber formation, but so far only a few fiber genes have been isolated and functionally characterized in this developmental process. The Kinesin13 subfamily was found to play key roles during cell division and cell elongation, and was considered to be involved in the regulation of cotton fiber development. The full length of coding sequence of GhKIS13A1 was cloned using cDNA from cotton fiber for functional characterization. Expression pattern analysis showed that GhKIS13A1 maintained a lower expression level during cotton fiber development. Biochemical assay showed that GhKIS13A1 has microtubule binding activity and basal ATPase activity that can be activated significantly by the presence of microtubules. Overexpression of GhKIS13A1 in Arabidopsis reduced leaf trichomes and the percentage of three-branch trichomes, and increased two-branch and shriveled trichomes compared to wild-type. Additionally, the expression of GhKIS13A1 in the Arabidopsis Kinesin-13a-1 mutant rescued the defective trichome branching pattern of the mutant, making its overall trichome branching pattern back to normal. Our results suggested that GhKIS13A1 is functionally compatible with AtKinesin-13A regarding their role in regulating the number and branching pattern of leaf trichomes. Given the developmental similarities between cotton fibers and Arabidopsis trichomes, it is speculated that GhKIS13A1 may also be involved in the regulation of cotton fiber development.

Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts

PubMed Central

1993-01-01

Xenopus egg extracts prepared before and after egg activation retain M- and S-phase specific activity, respectively. Staurosporine, a potent inhibitor of protein kinase, converted M-phase extracts into interphase- like extracts that were capable of forming nuclei upon the addition of sperm DNA. The nuclei formed in the staurosporine treated M-phase extract were incapable of replicating DNA, and they were unable to initiate replication upon the addition of S-phase extracts. Furthermore, replication was inhibited when the staurosporine-treated M- phase extract was added in excess to the staurosporine-treated S-phase extract before the addition of DNA. The membrane-depleted S-phase extract supported neither nuclear formation nor replication; however, preincubation of sperm DNA with these extracts allowed them to form replication-competent nuclei upon the addition of excess staurosporine- treated M-phase extract. These results demonstrate that positive factors in the S-phase extracts determined the initiation of DNA replication before nuclear formation, although these factors were unable to initiate replication after nuclear formation. PMID:8253833

Proteomic analysis of rodent ribosomes revealed heterogeneity including ribosomal proteins L10-like, L22-like 1, and L39-like.

PubMed

Sugihara, Yoshihiko; Honda, Hiroki; Iida, Tomoharu; Morinaga, Takuma; Hino, Shingo; Okajima, Tetsuya; Matsuda, Tsukasa; Nadano, Daita

2010-03-05

Heterogeneity of ribosome structure, due to variations in ribosomal protein composition, has been shown to be of physiological significance in plants and yeast. Mammalian genomics have demonstrated numerous genes that are paralogous to genes encoding ribosomal proteins. Although the vast majority are considered to be pseudogenes, mRNA expression of a few paralogues, such as human ribosomal protein L39-like/L39-2, has been reported. In the present study, ribosomes from the liver, mammary gland, and testis of rodents were analyzed using a combination of two-dimensional gel electrophoresis under radical-free and highly reducing conditions, and mass spectrometry. This system allowed identification of 78 ribosomal proteins and Rack1 from a single gel. The degree of heterogeneity was far less than that reported for plant and yeast ribosomes, and was in accord with published biochemical and genetic data for mammalian ribosomes. Nevertheless, an uncharacterized paralogue of ribosomal protein L22, ribosomal protein L22-like 1, was identified as a minor ribosomal component. Ribosomal proteins L10-like and L39-like, paralogues of ribosomal proteins L10 and L39, respectively, were found in ribosomes only from the testis. Reverse transcription-polymerase chain reaction yielded supportive evidence for specific expression of L10-like and L39-like in the testis. Newly synthesized L39-like is likely to be transported to the nucleolus, where ribosome biosynthesis occurs, and then incorporated into translating ribosomes in the cytoplasm. Heterogeneity of mammalian testicular ribosomes is structurally non-negligible, and may offer valuable insights into the function of the customized ribosome.

Light regulation of the abundance of mRNA encoding a nucleolin-like protein localized in the nucleoli of pea nuclei.

PubMed Central

Tong, C G; Reichler, S; Blumenthal, S; Balk, J; Hsieh, H L; Roux, S J

1997-01-01

A cDNA encoding a nucleolar protein was selected from a pea (Pisum sativum) plumule library, cloned, and sequenced. The translated sequence of the cDNA has significant percent identity to Xenopus laevis nucleolin (31%), the alfalfa (Medicago sativa) nucleolin homolog (66%), and the yeast (Saccharomyces cerevisiae) nucleolin homolog (NSR1) (28%). It also has sequence patterns in its primary structure that are characteristic of all nucleolins, including an N-terminal acidic motif, RNA recognition motifs, and a C-terminal Gly- and Arg-rich domain. By immunoblot analysis, the polyclonal antibodies used to select the cDNA bind selectively to a 90-kD protein in purified pea nuclei and nucleoli and to an 88-kD protein in extracts of Escherichia coli expressing the cDNA. In immunolocalization assays of pea plumule cells, the antibodies stained primarily a region surrounding the fibrillar center of nucleoli, where animal nucleolins are typically found. Southern analysis indicated that the pea nucleolin-like protein is encoded by a single gene, and northern analysis showed that the labeled cDNA binds to a single band of RNA, approximately the same size and the cDNA. After irradiation of etiolated pea seedlings by red light, the mRNA level in plumules decreased during the 1st hour and then increased to a peak of six times the 0-h level at 12 h. Far-red light reversed this effect of red light, and the mRNA accumulation from red/far-red light irradiation was equal to that found in the dark control. This indicates that phytochrome may regulate the expression of this gene. PMID:9193096

A smart dust biosensor powered by kinesin motors.

PubMed

Fischer, Thorsten; Agarwal, Ashutosh; Hess, Henry

2009-03-01

Biosensors can be miniaturized by either injecting smaller volumes into micro- and nanofluidic devices or immersing increasingly sophisticated particles known as 'smart dust' into the sample. The term 'smart dust' originally referred to cubic-millimetre wireless semiconducting sensor devices that could invisibly monitor the environment in buildings and public spaces, but later it also came to include functional micrometre-sized porous silicon particles used to monitor yet smaller environments. The principal challenge in designing smart dust biosensors is integrating transport functions with energy supply into the device. Here, we report a hybrid microdevice that is powered by ATP and relies on antibody-functionalized microtubules and kinesin motors to transport the target analyte into a detection region. The transport step replaces the wash step in traditional double-antibody sandwich assays. Owing to their small size and autonomous function, we envision that large numbers of such smart dust biosensors could be inserted into organisms or distributed into the environment for remote sensing.

A Matter of the Heart: The African Clawed Frog Xenopus as a Model for Studying Vertebrate Cardiogenesis and Congenital Heart Defects

PubMed Central

Hempel, Annemarie; Kühl, Michael

2016-01-01

The African clawed frog, Xenopus, is a valuable non-mammalian model organism to investigate vertebrate heart development and to explore the underlying molecular mechanisms of human congenital heart defects (CHDs). In this review, we outline the similarities between Xenopus and mammalian cardiogenesis, and provide an overview of well-studied cardiac genes in Xenopus, which have been associated with congenital heart conditions. Additionally, we highlight advantages of modeling candidate genes derived from genome wide association studies (GWAS) in Xenopus and discuss commonly used techniques. PMID:29367567

Klp10A modulates the localization of centriole-associated proteins during Drosophila male gametogenesis

PubMed Central

Gottardo, Marco; Callaini, Giuliano; Riparbelli, Maria Giovanna

2016-01-01

ABSTRACT Mutations in Klp10A, a microtubule-depolymerising Kinesin-13, lead to overly long centrioles in Drosophila male germ cells. We demonstrated that the loss of Klp10A modifies the distribution of typical proteins involved in centriole assembly and function. In the absence of Klp10A the distribution of Drosophila pericentrin-like protein (Dplp), Sas-4 and Sak/Plk4 that are restricted in control testes to the proximal end of the centriole increase along the centriole length. Remarkably, the cartwheel is lacking or it appears abnormal in mutant centrioles, suggesting that this structure may spatially delimit protein localization. Moreover, the parent centrioles that in control cells have the same dimensions grow at different rates in mutant testes with the mother centrioles longer than the daughters. Daughter centrioles have often an ectopic position with respect to the proximal end of the mothers and failed to recruit Dplp. PMID:27764551

Phospholipase C and D regulation of Src, calcium release and membrane fusion during Xenopus laevis development

PubMed Central

Stith, Bradley J.

2015-01-01

This review emphasizes how lipids regulate membrane fusion and the proteins involved in three developmental stages: oocyte maturation to the fertilizable egg, fertilization and during first cleavage. Decades of work show that phosphatidic acid (PA) releases intracellular calcium, and recent work shows that the lipid can activate Src tyrosine kinase or phospholipase C during Xenopus fertilization. Numerous reports are summarized to show three levels of increase in lipid second messengers inositol 1,4,5-trisphosphate and sn 1,2-diacylglycerol (DAG) during the three different developmental stages. In addition, possible roles for PA, ceramide, lysophosphatidylcholine, plasmalogens, phosphatidylinositol 4-phosphate, phosphatidylinositol 5-phosphate, phosphatidylinositol 4,5-bisphosphate, membrane microdomains (rafts) and phosphatidylinositol 3,4,5-trisphosphate in regulation of membrane fusion (acrosome reaction, sperm-egg fusion, cortical granule exocytosis), inositol 1,4,5-trisphosphate receptors, and calcium release are discussed. The role of six lipases involved in generating putative lipid second messengers during fertilization is also discussed: phospholipase D, autotaxin, lipin1, sphingomyelinase, phospholipase C, and phospholipase A2. More specifically, proteins involved in developmental events and their regulation through lipid binding to SH3, SH4, PH, PX, or C2 protein domains is emphasized. New models are presented for PA activation of Src (through SH3, SH4 and a unique domain), that this may be why the SH2 domain of PLCγ is not required for Xenopus fertilization, PA activation of phospholipase C, a role for PA during the calcium wave after fertilization, and that calcium/calmodulin may be responsible for the loss of Src from rafts after fertilization. Also discussed is that the large DAG increase during fertilization derives from phospholipase D production of PA and lipin dephosphorylation to DAG. PMID:25748412

Xenbase, the Xenopus model organism database; new virtualized system, data types and genomes.

PubMed

Karpinka, J Brad; Fortriede, Joshua D; Burns, Kevin A; James-Zorn, Christina; Ponferrada, Virgilio G; Lee, Jacqueline; Karimi, Kamran; Zorn, Aaron M; Vize, Peter D

2015-01-01

Xenbase (http://www.xenbase.org), the Xenopus frog model organism database, integrates a wide variety of data from this biomedical model genus. Two closely related species are represented: the allotetraploid Xenopus laevis that is widely used for microinjection and tissue explant-based protocols, and the diploid Xenopus tropicalis which is used for genetics and gene targeting. The two species are extremely similar and protocols, reagents and results from each species are often interchangeable. Xenbase imports, indexes, curates and manages data from both species; all of which are mapped via unique IDs and can be queried in either a species-specific or species agnostic manner. All our services have now migrated to a private cloud to achieve better performance and reliability. We have added new content, including providing full support for morpholino reagents, used to inhibit mRNA translation or splicing and binding to regulatory microRNAs. New genomes assembled by the JGI for both species and are displayed in Gbrowse and are also available for searches using BLAST. Researchers can easily navigate from genome content to gene page reports, literature, experimental reagents and many other features using hyperlinks. Xenbase has also greatly expanded image content for figures published in papers describing Xenopus research via PubMedCentral. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Analysis of 14-3-3 Family Member Function in Xenopus Embryos by Microinjection of Antisense Morpholino Oligos

NASA Astrophysics Data System (ADS)

Lau, Jeffrey M. C.; Muslin, Anthony J.

The 14-3-3 intracellular phosphoserine/threonine-binding proteins are adapter molecules that regulate signal transduction, cell cycle, nutrient sensing, apoptotic, and cytoskeletal pathways. There are seven 14-3-3 family members, encoded by separate genes, in vertebrate organisms. To evaluate the role of individual 14-3-3 proteins in vertebrate embryonic development, we utilized an antisense morpholino oligo microinjection technique in Xenopus laevis embryos. By use of this method, we showed that embryos lacking specific 14-3-3 proteins displayed unique phenotypic abnormalities. Specifically, embryos lacking 14-3-3 τ exhibited gastrulation and axial patterning defects, but embryos lacking 14-3-3 γ exhibited eye defects without other abnormalities, and embryos lacking 14-3-3 ζ appeared completely normal. These and other results demonstrate the power and specificity of the morpholino antisense oligo microinjection technique.

Heat shock proteins and toll-like receptors.

PubMed

Asea, Alexzander

2008-01-01

Researchers have only just begun to elucidate the relationship between heat shock proteins (HSP) and Toll-like receptors (TLR). HSP were originally described as an intracellular molecular chaperone of naïve, aberrantly folded, or mutated proteins and primarily implicated as a cytoprotective protein when cells are exposed to stressful stimuli. However, recent studies have ascribed novel functions to the Hsp70 protein depending on its localization: Surface-bound Hsp70 specifically activate natural killer (NK) cells, while Hsp70 released into the extracellular milieu specifically bind to Toll-like receptors (TLR) 2 and 4 on antigen-presenting cells (APC) and exerts immunoregulatory effects, including upregulation of adhesion molecules, co-stimulatory molecule expression, and cytokine and chemokine release-a process known as the chaperokine activity of Hsp70. This chapter discusses the most recent advances in the understanding of heat shock protein (HSP) and TLR interactions in general and highlights recent findings that demonstrate Hsp70 is a ligand for TLR and its biological significance.

Reproductive Maturation of the Tropical Clawed Frog, Xenopus tropicalis

EPA Science Inventory

The model species Xenopus tropicalis is being widely used in developmental biology and amphibian toxicology studies. In order to increase our understanding of the role of steroid hormones in maturation in this species, we collected baseline reproductive data from metamorphosis t...

Mammalian nuclear transplantation to Germinal Vesicle stage Xenopus oocytes – A method for quantitative transcriptional reprogramming

PubMed Central

Halley-Stott, R.P.; Pasque, V.; Astrand, C.; Miyamoto, K.; Simeoni, I.; Jullien, J.; Gurdon, J.B.

2010-01-01

Full-grown Xenopus oocytes in first meiotic prophase contain an immensely enlarged nucleus, the Germinal Vesicle (GV), that can be injected with several hundred somatic cell nuclei. When the nuclei of mammalian somatic cells or cultured cell lines are injected into a GV, a wide range of genes that are not transcribed in the donor cells, including pluripotency genes, start to be transcriptionally activated, and synthesize primary transcripts continuously for several days. Because of the large size and abundance of Xenopus laevis oocytes, this experimental system offers an opportunity to understand the mechanisms by which somatic cell nuclei can be reprogrammed to transcribe genes characteristic of oocytes and early embryos. The use of mammalian nuclei ensures that there is no background of endogenous maternal transcripts of the kind that are induced. The induced gene transcription takes place in the absence of cell division or DNA synthesis and does not require protein synthesis. Here we summarize new as well as established results that characterize this experimental system. In particular, we describe optimal conditions for transplanting somatic nuclei to oocytes and for the efficient activation of transcription by transplanted nuclei. We make a quantitative determination of transcript numbers for pluripotency and housekeeping genes, comparing cultured somatic cell nuclei with those of embryonic stem cells. Surprisingly we find that the transcriptional activation of somatic nuclei differs substantially from one donor cell-type to another and in respect of different pluripotency genes. We also determine the efficiency of an injected mRNA translation into protein. PMID:20123126

Insertional Mutagenesis for Genes involved in Otic/Vestibular Development and Function in Xenopus Tropicalis

NASA Technical Reports Server (NTRS)

Torrejon, Marcela; Li, Erica; Nguyen, Minh; Winfree, Seth; Wang, Esther; Reinsch, Sigrid; Dalton, Bonnie (Technical Monitor)

2002-01-01

Sensitivity to gravity is essential for spatial orientation. Consequently, the gravity receptor system is one of the phylogenetically oldest sensory systems, and the special adaptations that enhance sensitivity to gravity are highly conserved. The main goal of this project is to use Xenopus (frog) to identify genes expressed during vestibular and auditory development. These studies will lead a better understanding of the molecular mechanisms involved in vestibular and auditory development and function. We are using a gene-trap approach in Xenopus tropicalis with the green fluorescent protein (GFP) gene as the transgene reporter. GFP expression occurs only when the GFP gene is correctly integrated in actively transcribed genes. Using the GFP as a tag we can easily identify and clone the mutated gene. In addition, we can study the function of the mutated gene by analyzing the defects generated by insertion of the GFP transgene. To date we have tissue specific GFP expression in X. tropicalis including expression in ear, neural tube, kidney, muscle, eyes and nose. Our transgenic animals will soon reach maturity so that we can outcross them and analyze their progeny. Our next goal is to isolate RNA from our transgenics and clone the tagged genes using RACE-PCR. Currently we are optimizing the RACE-PCR method using transgenics with crystallin GFP expression.

Developing Xenopus Embryos Recover by Compacting and Expelling Single-Wall Carbon Nanotubes

PubMed Central

Holt, Brian D.; Shawky, Joseph H.; Dahl, Kris Noel; Davidson, Lance A.; Islam, Mohammad F.

2015-01-01

Single-wall carbon nanotubes are high aspect ratio nanomaterials that are being developed for use in materials, technological and biological applications due to their high mechanical stiffness, optical properties, and chemical inertness. Because of their prevalence, it is inevitable that biological systems will be exposed to nanotubes, yet studies of the effects of nanotubes on developing embryos have been inconclusive and are lacking for single-wall carbon nanotubes exposed to the widely studied model organism Xenopus laevis (African clawed frog). Microinjection of experimental substances into the Xenopus embryo is a standard technique for toxicology studies and cellular lineage tracing. Here we report the surprising finding that superficial (12.5 ± 7.5 μm below the membrane) microinjection of nanotubes dispersed with Pluronic F127 into one-to-two cell Xenopus embryos resulted in the formation and expulsion of compacted, nanotube-filled, punctate masses, at the blastula to mid-gastrula developmental stages, which we call “boluses”. Such expulsion of microinjected materials by Xenopus embryos has not been reported before and is dramatically different from the typical distribution of the materials throughout the progeny of the microinjected cells. Previous studies of microinjections of nanomaterials such as nanodiamonds, quantum dots or spherical nanoparticles report that nanomaterials often induce toxicity and remain localized within the embryos. In contrast, our results demonstrate an active recovery pathway for embryos after exposure to Pluronic F127-coated nanotubes, which we speculate is due to a combined effect of the membrane activity of the dispersing agent, Pluronic F127, and the large aspect ratio of nanotubes. PMID:26153061

Developing Xenopus embryos recover by compacting and expelling single wall carbon nanotubes.

PubMed

Holt, Brian D; Shawky, Joseph H; Dahl, Kris Noel; Davidson, Lance A; Islam, Mohammad F

2016-04-01

Single wall carbon nanotubes are high aspect ratio nanomaterials being developed for use in materials, technological and biological applications due to their high mechanical stiffness, optical properties and chemical inertness. Because of their prevalence, it is inevitable that biological systems will be exposed to nanotubes, yet studies of the effects of nanotubes on developing embryos have been inconclusive and are lacking for single wall carbon nanotubes exposed to the widely studied model organism Xenopus laevis (African clawed frog). Microinjection of experimental substances into the Xenopus embryo is a standard technique for toxicology studies and cellular lineage tracing. Here we report the surprising finding that superficial (12.5 ± 7.5 µm below the membrane) microinjection of nanotubes dispersed with Pluronic F127 into one- to two-cell Xenopus embryos resulted in the formation and expulsion of compacted, nanotube-filled, punctate masses, at the blastula to mid-gastrula developmental stages, which we call "boluses." Such expulsion of microinjected materials by Xenopus embryos has not been reported before and is dramatically different from the typical distribution of the materials throughout the progeny of the microinjected cells. Previous studies of microinjections of nanomaterials such as nanodiamonds, quantum dots or spherical nanoparticles report that nanomaterials often induce toxicity and remain localized within the embryos. In contrast, our results demonstrate an active recovery pathway for embryos after exposure to Pluronic F127-coated nanotubes, which we speculate is due to a combined effect of the membrane activity of the dispersing agent, Pluronic F127, and the large aspect ratio of nanotubes. Copyright © 2015 John Wiley & Sons, Ltd.

Intermolecular cleavage by UmuD-like mutagenesis proteins

PubMed Central

McDonald, John P.; Frank, Ekaterina G.; Levine, Arthur S.; Woodgate, Roger

1998-01-01

The activity of a number of proteins is regulated by self-processing reactions. Elegant examples are the cleavage of the prokaryotic LexA and λCI transcriptional repressors and the UmuD-like mutagenesis proteins. Various studies support the hypothesis that LexA and λCI cleavage reactions are predominantly intramolecular in nature. The recently described crystal structure of the Escherichia coli UmuD′ protein (the posttranslational cleavage product of the UmuD protein) suggests, however, that the region of the protein corresponding to the cleavage site is at least 50 Å away from the catalytic active site. We considered the possibility, therefore, that the UmuD-like proteins might undergo self-processing that, in contrast to LexA and λCI, occurs via an intermolecular rather than intramolecular reaction. To test this hypothesis, we introduced into E. coli compatible plasmids with mutations at either the cleavage or the catalytic site of three UmuD-like proteins. Cleavage of these proteins only occurs in the presence of both plasmids, indicating that the reaction is indeed intermolecular in nature. Furthermore, this intermolecular reaction is completely dependent upon the multifunctional RecA protein and leads to the restoration of cellular mutagenesis in nonmutable E. coli strains. Intermolecular cleavage of a biotinylated UmuD active site mutant was also observed in vitro in the presence of the wild-type UmuD′ protein, indicating that in addition to the intact UmuD protein, the normal cleavage product (UmuD′) can also act as a classical enzyme. PMID:9465040

Prolonged in vivo imaging of Xenopus laevis.

PubMed

Hamilton, Paul W; Henry, Jonathan J

2014-08-01

While live imaging of embryonic development over long periods of time is a well established method for embryos of the frog Xenopus laevis, once development has progressed to the swimming stages, continuous live imaging becomes more challenging because the tadpoles must be immobilized. Current imaging techniques for these advanced stages generally require bringing the tadpoles in and out of anesthesia for short imaging sessions at selected time points, severely limiting the resolution of the data. Here we demonstrate that creating a constant flow of diluted tricaine methanesulfonate (MS-222) over a tadpole greatly improves their survival under anesthesia. Based on this result, we describe a new method for imaging stage 48 to 65 X. laevis, by circulating the anesthetic using a peristaltic pump. This supports the animal during continuous live imaging sessions for at least 48 hr. The addition of a stable optical window allows for high quality imaging through the anesthetic solution. This automated imaging system provides for the first time a method for continuous observations of developmental and regenerative processes in advanced stages of Xenopus over 2 days. Developmental Dynamics 243:1011-1019, 2014. © 2014 Wiley Periodicals, Inc. © 2014 Wiley Periodicals, Inc.

LC3 binding to the scaffolding protein JIP1 regulates processive dynein-driven transport of autophagosomes.

PubMed

Fu, Meng-Meng; Nirschl, Jeffrey J; Holzbaur, Erika L F

2014-06-09

Autophagy is essential for maintaining cellular homeostasis in neurons, where autophagosomes undergo robust unidirectional retrograde transport along axons. We find that the motor scaffolding protein JIP1 binds directly to the autophagosome adaptor LC3 via a conserved LIR motif. This interaction is required for the initial exit of autophagosomes from the distal axon, for sustained retrograde transport along the midaxon, and for autophagosomal maturation in the proximal axon. JIP1 binds directly to the dynein activator dynactin but also binds to and activates kinesin-1 in a phosphorylation-dependent manner. Following JIP1 depletion, phosphodeficient JIP1-S421A rescues retrograde transport, while phosphomimetic JIP1-S421D aberrantly activates anterograde transport. During normal autophagosome transport, residue S421 of JIP1 may be maintained in a dephosphorylated state by autophagosome-associated MKP1 phosphatase. Moreover, binding of LC3 to JIP1 competitively disrupts JIP1-mediated activation of kinesin. Thus, dual mechanisms prevent aberrant activation of kinesin to ensure robust retrograde transport of autophagosomes along the axon. Copyright © 2014 Elsevier Inc. All rights reserved.

Experimental protocols for and studies of the effects of surface passivation and water isotopes on the gliding speed of microtubules propelled by kinesin-1

NASA Astrophysics Data System (ADS)

Maloney, Roger Andrew

This dissertation explores how the kinesin-1 and microtubule system is affected by surface passivation and water isotopes. Surface passivation was found to affect the gliding speed that microtubules exhibit in the gliding motility assay and the lengths of microtubules supported by the passivation. It was also found that gliding speeds of microtubules are very sensitive to temperature changes. Studies changing the water isotope were a first attempt to investigate if changing the solvent changed the osmotic pressure of the solution kinesin and microtubules were in. No osmotic pressure changes were observed, however, the experiments using different isotopes of water did illuminate the possibility that kinesin may be sensitive to viscosity changes in the solvent. This experiment also suggests further experiments that can be specifically designed to probe osmotic pressure changes. This thesis was also the first thesis ever, to the best of the author's knowledge, to be done in a completely open format. All information and notebook entries that are related to it, as well as the thesis itself, can be found on the website OpenWetWare. The thesis can also be found there including all the different versions that went into its editing. The philosophy and process of making data open and accessible to every one is also discussed.

Evidence that the rabbit proton-peptide co-transporter PepT1 is a multimer when expressed in Xenopus laevis oocytes.

PubMed

Panitsas, Konstantinos-E; Boyd, C A R; Meredith, David

2006-04-01

To test whether the rabbit proton-coupled peptide transporter PepT1 is a multimer, we have employed a combination of transport assays, luminometry and site-directed mutagenesis. A functional epitope-tagged PepT1 construct (PepT1-FLAG) was co-expressed in Xenopus laevis oocytes with a non-functional but normally trafficked mutant form of the same transporter (W294F-PepT1). The amount of PepT1-FLAG cRNA injected into the oocytes was kept constant, while the amount of W294F-PepT1 cRNA was increased over the mole fraction range of 0 to 1. The uptake of [(3)H]-D: -Phe-L: -Gln into the oocytes was measured at pH(out) 5.5, and the surface expression of PepT1-FLAG was quantified by luminometry. As the mole fraction of injected W294F-PepT1 increased, the uptake of D: -Phe-L: -Gln decreased. This occurred despite the surface expression of PepT1-FLAG remaining constant, and so we can conclude that PepT1 must be a multimer. Assuming that PepT1 acts as a homomultimer, the best fit for the modelling suggests that PepT1 could be a tetramer, with a minimum requirement of two functional subunits in each protein complex. Western blotting also showed the presence of higher-order complexes of PepT1-FLAG in oocyte membranes. It should be noted that we cannot formally exclude the possibility that PepT1 interacts with unidentified Xenopus protein(s). The finding that PepT1 is a multimer has important implications for the molecular modelling of this protein.

Expression of the mammalian calcium signaling response to Trypanosoma cruzi in Xenopus laevis oocytes.

PubMed

Leite, M F; Moyer, M S; Andrews, N W

1998-04-01

Infective stages of the protozoan parasite Trypanosoma cruzi contain a soluble factor that induces elevation in the intracellular free Ca2+ concentration ([Ca2+]i) of mammalian cells. The process is pertussis toxin (PTx)-sensitive, and involves phospholipase C (PLC) activation, inositol 1,4,5-trisphosphate (IP3) formation and Ca2+ release from intracellular stores (Tardieux I, et al. J Exp Med 1994;179:1017-1022; Rodriguez A, et al. J Cell Biol 1995;129:1263-1273). We now report that a molecule exposed on the surface of the target cells is required to trigger the signaling cascade, and that a response with identical characteristics can be induced in Xenopus laevis oocytes injected with mRNA from normal rat kidney (NRK) fibroblasts. Xenopus oocytes do not show an endogenous response to the trypomastigote Ca2+ signaling factor, but a vigorous response in the form of a propagating Ca2+ wave is expressed after injection of NRK cell mRNA. As previously demonstrated for mammalian cells, the response is inhibited when injected oocytes are pretreated with PTx, implicating Galphai or Galphao trimeric G-proteins, and with thapsigargin, which depletes intracellular Ca2+ stores. Moreover, the [Ca2+]i transients triggered by the T. cruzi soluble factor in mRNA-injected oocytes are blocked by the same inhibitors of the parasite oligopeptidase B that abolish the [Ca2+]i response in NRK cells (Burleigh B, Andrews NW. J Biol Chem 1995;270:5172-5180; Burleigh BA et al. J Cell Biol 1997;136:609-620). The NRK mRNA fraction that induces expression of the [Ca2+]i response to the T. cruzi signaling factor contains messages from 1.5 to 2.0 kb, a size range consistent with the family of seven-transmembrane G-protein-coupled receptors.

The microtubule motor protein KIF13A is involved in intracellular trafficking of the Lassa virus matrix protein Z.

PubMed

Fehling, Sarah Katharina; Noda, Takeshi; Maisner, Andrea; Lamp, Boris; Conzelmann, Karl-Klaus; Kawaoka, Yoshihiro; Klenk, Hans-Dieter; Garten, Wolfgang; Strecker, Thomas

2013-02-01

The small matrix protein Z of arenaviruses has been identified as the main driving force to promote viral particle production at the plasma membrane. Although multiple functions of Z in the arenaviral life cycle have been uncovered, the mechanism of intracellular transport of Z to the site of virus budding is poorly understood and cellular motor proteins that mediate Z trafficking remain to be identified. In the present study, we report that the Z protein of the Old World arenavirus Lassa virus (LASV) interacts with the kinesin family member 13A (KIF13A), a plus-end-directed microtubule-dependent motor protein. Plasmid-driven overexpression of KIF13A results in relocalization of Z to the cell periphery, while functional blockage of endogenous KIF13A by overexpression of a dominant-negative mutant or KIF13A-specific siRNA causes a perinuclearaccumulation and decreased production of both Z-induced virus-like particles and infectious LASV. The interaction of KIF13A with Z proteins from both Old and New World arenaviruses suggests a conserved intracellular transport mechanism. In contrast, the intracellular distribution of the matrix proteins of prototypic members of the paramyxo- and rhabdovirus family is independent of KIF13A. In summary, our studies identify for the first time a molecular motor protein as a critical mediator for intracellular microtubule-dependent transport of arenavirus matrix proteins. © 2012 Blackwell Publishing Ltd.

Evolutionary hierarchy of vertebrate-like heterotrimeric G protein families.

PubMed

Krishnan, Arunkumar; Mustafa, Arshi; Almén, Markus Sällman; Fredriksson, Robert; Williams, Michael J; Schiöth, Helgi B

2015-10-01

Heterotrimeric G proteins perform a crucial role as molecular switches controlling various cellular responses mediated by G protein-coupled receptor (GPCR) signaling pathway. Recent data have shown that the vertebrate-like G protein families are found across metazoans and their closest unicellular relatives. However, an overall evolutionary hierarchy of vertebrate-like G proteins, including gene family annotations and in particular mapping individual gene gain/loss events across diverse holozoan lineages is still incomplete. Here, with more expanded invertebrate taxon sampling, we have reconstructed phylogenetic trees for each of the G protein classes/families and provide a robust classification and hierarchy of vertebrate-like heterotrimeric G proteins. Our results further extend the evidence that the common ancestor (CA) of holozoans had at least five ancestral Gα genes corresponding to all major vertebrate Gα classes and contain a total of eight genes including two Gβ and one Gγ. Our results also indicate that the GNAI/O-like gene likely duplicated in the last CA of metazoans to give rise to GNAI- and GNAO-like genes, which are conserved across invertebrates. Moreover, homologs of GNB1-4 paralogon- and GNB5 family-like genes are found in most metazoans and that the unicellular holozoans encode two ancestral Gβ genes. Similarly, most bilaterian invertebrates encode two Gγ genes which include a representative of the GNG gene cluster and a putative homolog of GNG13. Interestingly, our results also revealed key evolutionary events such as the Drosophila melanogaster eye specific Gβ subunit that is found conserved in most arthropods and several previously unidentified species specific expansions within Gαi/o, Gαs, Gαq, Gα12/13 classes and the GNB1-4 paralogon. Also, we provide an overall proposed evolutionary scenario on the expansions of all G protein families in vertebrate tetraploidizations. Our robust classification/hierarchy is essential to further

Characterization of Cer-1 cis-regulatory region during early Xenopus development.

PubMed

Silva, Ana Cristina; Filipe, Mário; Steinbeisser, Herbert; Belo, José António

2011-05-01

Cerberus-related molecules are well-known Wnt, Nodal, and BMP inhibitors that have been implicated in different processes including anterior–posterior patterning and left–right asymmetry. In both mouse and frog, two Cerberus-related genes have been isolated, mCer-1 and mCer-2, and Xcer and Xcoco, respectively. Until now, little is known about the mechanisms involved in their transcriptional regulation. Here, we report a heterologous analysis of the mouse Cerberus-1 gene upstream regulatory regions, responsible for its expression in the visceral endodermal cells. Our analysis showed that the consensus sequences for a TATA, CAAT, or GC boxes were absent but a TGTGG sequence was present at position -172 to -168 bp, relative to the ATG. Using a series of deletion constructs and transient expression in Xenopus embryos, we found that a fragment of 1.4 kb of Cer-1 promoter sequence could reproduce the endogenous expression pattern of Xenopus cerberus. A 0.7-kb mcer-1 upstream region was able to drive reporter expression to the involuting mesendodermal cells, while further deletions abolished reporter gene expression. Our results suggest that although no sequence similarity was found between mouse and Xenopus cerberus cis-regulatory regions, the signaling cascades regulating cerberus expression, during gastrulation, is conserved.

The Midblastula Transition Defines the Onset of Y RNA-Dependent DNA Replication in Xenopus laevis ▿

PubMed Central

Collart, Clara; Christov, Christo P.; Smith, James C.; Krude, Torsten

2011-01-01

Noncoding Y RNAs are essential for the initiation of chromosomal DNA replication in mammalian cell extracts, but their role in this process during early vertebrate development is unknown. Here, we use antisense morpholino nucleotides (MOs) to investigate Y RNA function in Xenopus laevis and zebrafish embryos. We show that embryos in which Y RNA function is inhibited by MOs develop normally until the midblastula transition (MBT) but then fail to replicate their DNA and die before gastrulation. Consistent with this observation, Y RNA function is not required for DNA replication in Xenopus egg extracts but is required for replication in a post-MBT cell line. Y RNAs do not bind chromatin in karyomeres before MBT, but they associate with interphase nuclei after MBT in an origin recognition complex (ORC)-dependent manner. Y RNA-specific MOs inhibit the association of Y RNAs with ORC, Cdt1, and HMGA1a proteins, suggesting that these molecular associations are essential for Y RNA function in DNA replication. The MBT is thus a transition point between Y RNA-independent and Y RNA-dependent control of vertebrate DNA replication. Our data suggest that in vertebrates Y RNAs function as a developmentally regulated layer of control over the evolutionarily conserved eukaryotic DNA replication machinery. PMID:21791613

Probing intracellular motor protein activity using an inducible cargo trafficking assay.

PubMed

Kapitein, Lukas C; Schlager, Max A; van der Zwan, Wouter A; Wulf, Phebe S; Keijzer, Nanda; Hoogenraad, Casper C

2010-10-06

Although purified cytoskeletal motor proteins have been studied extensively with the use of in vitro approaches, a generic approach to selectively probe actin and microtubule-based motor protein activity inside living cells is lacking. To examine specific motor activity inside living cells, we utilized the FKBP-rapalog-FRB heterodimerization system to develop an in vivo peroxisomal trafficking assay that allows inducible recruitment of exogenous and endogenous kinesin, dynein, and myosin motors to drive specific cargo transport. We demonstrate that cargo rapidly redistributes with distinct dynamics for each respective motor, and that combined (antagonistic) actions of more complex motor combinations can also be probed. Of importance, robust cargo redistribution is readily achieved by one type of motor protein and does not require the presence of opposite-polarity motors. Simultaneous live-cell imaging of microtubules and kinesin or dynein-propelled peroxisomes, combined with high-resolution particle tracking, revealed that peroxisomes frequently pause at microtubule intersections. Titration and washout experiments furthermore revealed that motor recruitment by rapalog-induced heterodimerization is dose-dependent but irreversible. Our assay directly demonstrates that robust cargo motility does not require the presence of opposite-polarity motors, and can therefore be used to characterize the motile properties of specific types of motor proteins. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Xenopus-derived glucagon-like peptide-1 and polyethylene-glycosylated glucagon-like peptide-1 receptor agonists: long-acting hypoglycaemic and insulinotropic activities with potential therapeutic utilities.

PubMed

Han, Jing; Fei, Yingying; Zhou, Feng; Chen, Xinyu; Zhang, Ying; Liu, Lin; Fu, Junjie

2018-02-01

Incretin-based therapies based on glucagon-like peptide-1 (GLP-1) receptor agonists are effective treatments of type 2 diabetes. Abundant research has focused on the development of long-acting GLP-1 receptor agonists. However, all GLP-1 receptor agonists in clinical use or development are based on human or Gila GLP-1. We have identified a potent GLP-1 receptor agonist, xGLP-1B, based on Xenopus GLP-1. To further modify the structure of xGLP-1B, alanine scanning was performed to study the structure -activity relationship of xGLP-1B. Two strategies were then employed to improve bioactivity. First, the C-terminal tail of lixisenatide was appended to cysteine-altered xGLP-1B analogues. Second, polyethylene glycol (PEG) chains with different molecular weights were conjugated with the peptides, giving a series of PEGylated conjugates. Comprehensive bioactivity studies of these conjugates were performed in vitro and in vivo. From the in vitro receptor activation potency and in vivo acute hypoglycaemic activities of conjugates 25 -36, 33 was identified as the best candidate for further biological assessments. Conjugate 33 exhibited prominent hypoglycaemic and insulinotropic activities, as well as improved pharmacokinetic profiles in vivo. The prolonged antidiabetic duration of 33 was further confirmed by pre-oral glucose tolerance tests (OGTT) and multiple OGTT. Furthermore, chronic treatment of db/db mice with 33 ameliorated non-fasting blood glucose and insulin levels, reduced HbA1c values and normalized their impaired glucose tolerance. Importantly, no in vivo toxicity was observed in mice treated with 33. Peptide 33 is a promising long-acting type 2 diabetes therapeutic deserving further investigation. © 2017 The British Pharmacological Society.

Collagen-Like Proteins in Pathogenic E. coli Strains

PubMed Central

Ghosh, Neelanjana; McKillop, Thomas J.; Jowitt, Thomas A.; Howard, Marjorie; Davies, Heather; Holmes, David F.; Roberts, Ian S.; Bella, Jordi

2012-01-01

The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages. PMID:22701585

Nucleosome Translational Position, Not Histone Acetylation, Determines TFIIIA Binding to Nucleosomal Xenopus laevis 5S rRNA Genes

PubMed Central

Howe, LeAnn; Ausió, Juan

1998-01-01

We sought to study the binding constraints placed on the nine-zinc-finger protein transcription factor IIIA (TFIIIA) by a histone octamer. To this end, five overlapping fragments of the Xenopus laevis oocyte and somatic 5S rRNA genes were reconstituted into nucleosomes, and it was subsequently shown that nucleosome translational positioning is a major determinant of the binding of TFIIIA to the 5S rRNA genes. Furthermore, it was found that histone acetylation cannot override the TFIIIA binding constraints imposed by unfavorable translational positions. PMID:9488430

Mechanism-based Proteomic Screening Identifies Targets of Thioredoxin-like Proteins*

PubMed Central

Nakao, Lia S.; Everley, Robert A.; Marino, Stefano M.; Lo, Sze M.; de Souza, Luiz E.; Gygi, Steven P.; Gladyshev, Vadim N.

2015-01-01

Thioredoxin (Trx)-fold proteins are protagonists of numerous cellular pathways that are subject to thiol-based redox control. The best characterized regulator of thiols in proteins is Trx1 itself, which together with thioredoxin reductase 1 (TR1) and peroxiredoxins (Prxs) comprises a key redox regulatory system in mammalian cells. However, there are numerous other Trx-like proteins, whose functions and redox interactors are unknown. It is also unclear if the principles of Trx1-based redox control apply to these proteins. Here, we employed a proteomic strategy to four Trx-like proteins containing CXXC motifs, namely Trx1, Rdx12, Trx-like protein 1 (Txnl1) and nucleoredoxin 1 (Nrx1), whose cellular targets were trapped in vivo using mutant Trx-like proteins, under conditions of low endogenous expression of these proteins. Prxs were detected as key redox targets of Trx1, but this approach also supported the detection of TR1, which is the Trx1 reductant, as well as mitochondrial intermembrane proteins AIF and Mia40. In addition, glutathione peroxidase 4 was found to be a Rdx12 redox target. In contrast, no redox targets of Txnl1 and Nrx1 could be detected, suggesting that their CXXC motifs do not engage in mixed disulfides with cellular proteins. For some Trx-like proteins, the method allowed distinguishing redox and non-redox interactions. Parallel, comparative analyses of multiple thiol oxidoreductases revealed differences in the functions of their CXXC motifs, providing important insights into thiol-based redox control of cellular processes. PMID:25561728

Xenopus in Space and Time: Fossils, Node Calibrations, Tip-Dating, and Paleobiogeography.

PubMed

Cannatella, David

2015-01-01

Published data from DNA sequences, morphology of 11 extant and 15 extinct frog taxa, and stratigraphic ranges of fossils were integrated to open a window into the deep-time evolution of Xenopus. The ages and morphological characters of fossils were used as independent datasets to calibrate a chronogram. We found that DNA sequences, either alone or in combination with morphological data and fossils, tended to support a close relationship between Xenopus and Hymenochirus, although in some analyses this topology was not significantly better than the Pipa + Hymenochirus topology. Analyses that excluded DNA data found strong support for the Pipa + Hymenochirus tree. The criterion for selecting the maximum age of the calibration prior influenced the age estimates, and our age estimates of early divergences in the tree of frogs are substantially younger than those of published studies. Node-dating and tip-dating calibrations, either alone or in combination, yielded older dates for nodes than did a root calibration alone. Our estimates of divergence times indicate that overwater dispersal, rather than vicariance due to the splitting of Africa and South America, may explain the presence of Xenopus in Africa and its closest fossil relatives in South America.

Confocal Imaging of Early Heart Development in Xenopus laevis

PubMed Central

Kolker, Sandra J.; Tajchman, Urszula; Weeks, Daniel L.

2013-01-01

Xenopus laevis provides a number of advantages for studies on cardiovascular development. The embryos are fairly large, easy to obtain, and can develop at ambient temperature in simple buffer solutions. Although classic descriptions of heart development exist, the ability to use whole mount immunohistochemical methods and confocal microscopy may enhance the ability to understand both normal and experimentally perturbed cardiovascular development. We have started to examine the early stages of cardiac development in Xenopus, seeking to identify antibodies and fixatives that allow easy examination of the developing heart. We have used monoclonal antibodies (mAbs) raised against bovine cardiac troponin T and chicken tropomyosin to visualize cardiac muscle, a goat antibody recognizing bovine type VI collagen to stain the lining of vessels, and the JB3 mAb raised against chicken fibrillin which allows the visualization of a variety of cardiovascular tissues during early development. Results from embryonic stages 24–46 are presented. PMID:10644411

Characterization of Pax3 and Sox10 transgenic Xenopus laevis embryos as tools to study neural crest development.

PubMed

Alkobtawi, Mansour; Ray, Heather; Barriga, Elias H; Moreno, Mauricio; Kerney, Ryan; Monsoro-Burq, Anne-Helene; Saint-Jeannet, Jean-Pierre; Mayor, Roberto

2018-03-06

The neural crest is a multipotent population of cells that originates a variety of cell types. Many animal models are used to study neural crest induction, migration and differentiation, with amphibians and birds being the most widely used systems. A major technological advance to study neural crest development in mouse, chick and zebrafish has been the generation of transgenic animals in which neural crest specific enhancers/promoters drive the expression of either fluorescent proteins for use as lineage tracers, or modified genes for use in functional studies. Unfortunately, no such transgenic animals currently exist for the amphibians Xenopus laevis and tropicalis, key model systems for studying neural crest development. Here we describe the generation and characterization of two transgenic Xenopus laevis lines, Pax3-GFP and Sox10-GFP, in which GFP is expressed in the pre-migratory and migratory neural crest, respectively. We show that Pax3-GFP could be a powerful tool to study neural crest induction, whereas Sox10-GFP could be used in the study of neural crest migration in living embryos. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

A Coarse-Grained Protein Model in a Water-like Solvent

NASA Astrophysics Data System (ADS)

Sharma, Sumit; Kumar, Sanat K.; Buldyrev, Sergey V.; Debenedetti, Pablo G.; Rossky, Peter J.; Stanley, H. Eugene

2013-05-01

Simulations employing an explicit atom description of proteins in solvent can be computationally expensive. On the other hand, coarse-grained protein models in implicit solvent miss essential features of the hydrophobic effect, especially its temperature dependence, and have limited ability to capture the kinetics of protein folding. We propose a free space two-letter protein (``H-P'') model in a simple, but qualitatively accurate description for water, the Jagla model, which coarse-grains water into an isotropically interacting sphere. Using Monte Carlo simulations, we design protein-like sequences that can undergo a collapse, exposing the ``Jagla-philic'' monomers to the solvent, while maintaining a ``hydrophobic'' core. This protein-like model manifests heat and cold denaturation in a manner that is reminiscent of proteins. While this protein-like model lacks the details that would introduce secondary structure formation, we believe that these ideas represent a first step in developing a useful, but computationally expedient, means of modeling proteins.

Does acute or habitual protein deprivation influence liking for monosodium glutamate?

PubMed

Masic, Una; Yeomans, Martin R

2017-03-15

The umami flavour generated by monosodium glutamate (MSG) has been proposed as the marker for the presence of protein in foods. As protein is the most closely regulated macronutrient in the diet, the present study addressed whether acute protein deprivation, habitual protein intake or a combination of the two influenced liking for the taste of MSG. 24 low-restraint male participants (mean age: 22; BMI: 23) consumed either their habitual breakfast (baseline), a low protein breakfast (breakfast meal with low protein milk and milkshake) or a high protein breakfast (breakfast meal with high protein milk and milkshake) on three different days, and then evaluated the acceptability of umami (MSG), salty (NaCl) or sweet (Acesulphame K) tastes at low or high concentrations in a soup context at lunchtime. Participants also completed a habitual protein intake questionnaire (39-item protein Food Frequency Questionnaire). Liking for all tastes was higher on the low than on the high protein day, and NaCl and Acesulphame K were liked less on both protein manipulation days when compared to the no added flavour control. Habitual protein intake was not related to liking for MSG stimuli alone but habitual high protein consumers rated a high concentration of MSG as more pleasant than any other taste when in protein deficit. Overall, these findings suggest that liking for high MSG concentrations may be moderated by nutritional need in high protein consumers. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

Evaluation of microtransplantation of rat brain neurolemma into Xenopus laevis oocytes as a technique to study the effect of neurotoxicants on endogenous voltage-sensitive ion channels.

PubMed

Murenzi, Edwin; Toltin, Abigail C; Symington, Steven B; Morgan, Molly M; Clark, John M

2017-05-01

Microtransplantation of mammalian brain neurolemma into the plasma membrane of Xenopus oocytes is used to study ion channels in their native form as they appear in the central nervous system. Use of microtransplanted neurolemma is advantageous for various reasons: tissue can be obtained from various sources and at different developmental stages; ion channels and receptors are present in their native configuration in their proper lipid environment along with appropriate auxiliary subunits; allowing the evaluation of numerous channelpathies caused by neurotoxicants in an ex vivo state. Here we show that Xenopus oocytes injected with post-natal day 90 (PND90) rat brain neurolemma fragments successfully express functional ion channels. Using a high throughput two electrode voltage clamp (TEVC) electrophysiological system, currents that were sensitive to tetrodotoxin, ω-conotoxin MVIIC, and tetraethylammonium were detected, indicating the presence of multiple voltage-sensitive ion channels (voltage-sensitive sodium (VSSC), calcium and potassium channels, respectively). The protein expression pattern for nine different VSSC isoforms (Na v 1.1-Na v 1.9) was determined in neurolemma using automated western blotting, with the predominant isoforms expressed being Na v 1.2 and Na v 1.6. VSSC were also successfully detected in the plasma membrane of Xenopus oocytes microtransplanted with neurolemma. Using this approach, a "proof-of-principle" experiment was conducted where a well-established structure-activity relationship between the neurotoxicant, 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT) and its non-neurotoxic metabolite, 1,1-bis-(4-chlorophenyl)-2,2-dichloroethene (DDE) was examined. A differential sensitivity of DDT and DDE on neurolemma-injected oocytes was determined where DDT elicited a concentration-dependent increase in TTX-sensitive inward sodium current upon pulse-depolarization whereas DDE resulted in no significant effect. Additionally, DDT resulted in

Molecular characterization and analysis of a novel protein disulfide isomerase-like protein of Eimeria tenella.

PubMed

Han, Hongyu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Jiang, Lianlian; Wang, Yange; Li, Liujia; Wu, Youlin; Huang, Bing

2014-01-01

Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC). BLAST analysis showed that the EtPDIL protein was 55-59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells. These results

Molecular Characterization and Analysis of a Novel Protein Disulfide Isomerase-Like Protein of Eimeria tenella

PubMed Central

Han, Hongyu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Jiang, Lianlian; Wang, Yange; Li, Liujia; Wu, Youlin; Huang, Bing

2014-01-01

Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC). BLAST analysis showed that the EtPDIL protein was 55–59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells. These results

Further Characterization of an Interleukin-2-1Ike Cytokine Produced by Xenopus Laevis T Lymphocytes

PubMed Central

Haynes, Laura

1993-01-01

A T-cell growth factor (TCGF) is produced by antigen- or mitogen-stimulated T lymphocytes from the South African clawed frog Xenopus laevis. This study further defines the physical and biological properties of this cytokine and demonstrates that TCGF is biochemically similar to mammalian interleukin-2 (IL-2). Biologically active TCGF eluted from SDS-PAGE displays a Mr of 16 kD and lectin-affinity chromatography indicates that the three-dimensionmal configuration of carbohydrates on TCGF and human IL-2 is similar. Secretion of TCGF is detectable 1 day after stimulation of splenocytes with the T-cell mitogen phytohemagglutinin (PHA) and peaks following 2 to 3 days of stimulation. Finally, despite the biological and physical similarities between Xenopus TCGF and mammalian IL-2, anti-human IL-2 monoclonal antibodies do not recognize Xenopus TCGF. PMID:8281036

Acid-induced off-response of PKD2L1 channel in Xenopus oocytes and its regulation by Ca2+

PubMed Central

Hussein, Shaimaa; Zheng, Wang; Dyte, Chris; Wang, Qian; Yang, JungWoo; Zhang, Fan; Tang, Jingfeng; Cao, Ying; Chen, Xing-Zhen

2015-01-01

Polycystic kidney disease (PKD) protein 2 Like 1 (PKD2L1), also called transient receptor potential polycystin-3 (TRPP3), regulates Ca2+-dependent hedgehog signalling in primary cilia, intestinal development and sour tasting but with an unclear mechanism. PKD2L1 is a Ca2+-permeable cation channel that is activated by extracellular Ca2+ (on-response) in Xenopus oocytes. PKD2L1 co-expressed with PKD protein 1 Like 3 (PKD1L3) exhibits extracellular acid-induced activation (off-response, i.e., activation following acid removal) but whether PKD1L3 participates in acid sensing remains unclear. Here we used the two-microelectrode voltage-clamp, site directed mutagenesis, Western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence, and showed that PKD2L1 expressed in oocytes exhibits sustained off-response currents in the absence of PKD1L3. PKD1L3 co-expression augmented the PKD2L1 plasma membrane localization but did not alter the observed properties of the off-response. PKD2L1 off-response was inhibited by an increase in intracellular Ca2+. We also identified two intra-membrane residues aspartic acid 349 (D349) and glutamic acid 356 (E356) in the third transmembrane domain that are critical for PKD2L1 channel function. Our study suggests that PKD2L1 may itself sense acids and defines off-response properties in the absence of PKD1L3. PMID:26502994

Host-defense and trefoil factor family peptides in skin secretions of the Mawa clawed frog Xenopus boumbaensis (Pipidae).

PubMed

Conlon, J Michael; Mechkarska, Milena; Kolodziejek, Jolanta; Leprince, Jérôme; Coquet, Laurent; Jouenne, Thierry; Vaudry, Hubert; Nowotny, Norbert; King, Jay D

2015-10-01

Peptidomic analysis of norepinephrine-stimulated skin secretions from the octoploid Mawa clawed frog Xenopus boumbaensis Loumont, 1983 led to the identification and characterization of 15 host-defense peptides belonging to the magainin (two peptides), peptide glycine-leucine-amide (PGLa; three peptides), xenopsin precursor fragment (XPF; three peptides), caerulein precursor fragment (CPF; two peptides), and caerulein precursor fragment-related peptide (CPF-RP; five peptides) families. In addition, caerulein and three peptides with structural similarity to the trefoil factor family (TFF) peptides, xP2 and xP4 from Xenopus laevis were also present in the secretions. Consistent with data from comparisons of the nucleotides sequence of mitochondrial and nuclear genes, the primary structures of the peptides suggest a close phylogenetic relationship between X. boumbaensis and the octoploid frogs Xenopus amieti and Xenopus andrei. As the three species occupy disjunct ranges within Cameroon, it is suggested that they diverged from a common ancestor by allopatric speciation. Copyright © 2015 Elsevier Inc. All rights reserved.

Regulative development of Xenopus laevis in microgravity