E-MAP-115 (ensconsin) associates dynamically with microtubules in vivo and is not a physiological modulator of microtubule dynamics

Microtubule-associated proteins (MAPs) have been hypothesized to regulate microtubule dynamics and/or functions. To test hypotheses concerning E-MAP-115 (ensconsin) function, we prepared stable cell lines expressing conjugates in which the full-length MAP (Ensc) or its microtubule-binding domain (EMTB) was conjugated to one or more green fluorescent protein (GFP) molecules. Because both distribution and microtubule-binding properties of GFP-Ensc, GFP-EMTB, and 2x, 3x, or 4xGFP-EMTB chimeras all appeared to be identical to those of endogenous E-MAP-115 (ensconsin), we used the 2xGFP-EMTB molecule as a reporter for the behavior and microtubule-binding function of endogenous MAP. Dual wavelength time-lapse fluorescence imaging of 2xGFP-EMTB in cells microinjected with labeled tubulin revealed that this GFP-MAP chimera associated with the lattice of all microtubules immediately upon polymerization and dissociated concomitant with depolymerization, suggesting that dynamics of MAP:microtubule interactions were at least as rapid as tubulin:microtubule dynamics in the polymerization reaction. Presence of both GFP-EMTB chimeras and endogenous E-MAP-115 (ensconsin) along apparently all cellular microtubules at all cell cycle stages suggested that the MAP might function in modulating stability or dynamics of microtubules, a capability shown previously in transiently transfected cells. Although cells with extremely high expression levels of GFP-EMTB chimera exhibited stabilized microtubules, cells expressing four to ten times the physiological level of endogenous MAP exhibited microtubule dynamics indistinguishable from those of untransfected cells. This result shows that E-MAP-115 (ensconsin) is unlikely to function as a microtubule stabilizer in vivo. Instead, this MAP most likely serves to modulate microtubule functions or interactions with other cytoskeletal elements.

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Rapid dynamics of the microtubule binding of ensconsin in vivo

Journal of Cell Science ◽

10.1242/jcs.114.21.3885 ◽

2001 ◽

Vol 114 (21) ◽

pp. 3885-3897 ◽

Cited By ~ 3

Author(s):

J. Chloë Bulinski ◽

David J. Odde ◽

Bonnie J. Howell ◽

Ted D. Salmon ◽

Clare M. Waterman-Storer

Keyword(s):

Fluorescent Protein ◽

Living Cells ◽

Microtubule Dynamics ◽

Full Length ◽

Half Time ◽

Microtubule Binding ◽

Associated Proteins ◽

Green Fluorescent

Microtubule-associated proteins (MAPs) are proteins that reversibly bind to and regulate microtubule dynamics and functions in vivo. We examined the dynamics of binding of a MAP called ensconsin (E-MAP-115) to microtubules in vivo. We used 5×GFP-EMTB, a construct in which the microtubule-binding domain of ensconsin (EMTB) is fused to five copies of green fluorescent protein (GFP), as a reporter molecule amenable to the use of fluorescent speckle microscopy. Fluorescent speckle microscopy (FSM) sequences and kymograph analyses showed rapid dynamics of speckles comprised of 5×GFP-EMTB in untreated cells. By contrast, in detergent-lysed cytoskeletons, speckles were not dynamic. Since detergent-lysed cytoskeletons differ from living cells in that they lack both ATP and dynamic microtubules, we used azide treatment to substantially reduce the level of ATP in living cells and we used Taxol to halt microtubule dynamics. Both treatments slowed the dynamics of 5×GFP-EMTB speckles observed by FSM. We also used fluorescence recovery after photobleaching (FRAP) to quantify the half-time of binding and dissociation of the 5×GFP-EMTB chimera and to compare this half-time to that of the full-length MAP molecule. In untreated cells, the tg of either 5×GFP-EMTB or full-length GFP-ensconsin was similarly rapid (∼4 seconds), while in ATP-reduced and Taxol-treated cells, tg was increased to 210 seconds and 40 seconds, respectively. In detergent-extracted cells no recovery was seen. Consistent with the rapid dynamics of 5×GFP-EMTB measured with fluorescent speckle microscopy and FRAP, we estimated that the affinity of the MAP for microtubules is ∼40 μM in untreated living cells, compared with ∼1 μM in vitro. However, KD,app was not significantly changed in the presence of azide and was increased to 110 μM in the presence of Taxol. To test whether changes in the phosphorylation state of cellular proteins might be responsible for altering the dynamics of ensconsin binding, we used FSM to monitor staurosporine-treated cells. Staurosporine treatment substantially halted dynamics of 5×GFP-EMTB speckles along MTs. Our results show that ensconsin is highly dynamic in its association with microtubules, and its microtubule association can be altered by in vivo phosphorylation events.

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Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells

Blood ◽

10.1182/blood-2010-01-264382 ◽

2010 ◽

Vol 116 (6) ◽

pp. 909-914 ◽

Cited By ~ 114

Author(s):

Enid Yi Ni Lam ◽

Christopher J. Hall ◽

Philip S. Crosier ◽

Kathryn E. Crosier ◽

Maria Vega Flores

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Fluorescent Protein ◽

Living Organism ◽

Time Lapse ◽

Dorsal Aorta ◽

Transgenic Zebrafish ◽

Hematopoietic Stem ◽

Green Fluorescent

Abstract Blood cells of an adult vertebrate are continuously generated by hematopoietic stem cells (HSCs) that originate during embryonic life within the aorta-gonad-mesonephros region. There is now compelling in vivo evidence that HSCs are generated from aortic endothelial cells and that this process is critically regulated by the transcription factor Runx1. By time-lapse microscopy of Runx1-enhanced green fluorescent protein transgenic zebrafish embryos, we were able to capture a subset of cells within the ventral endothelium of the dorsal aorta, as they acquire hemogenic properties and directly emerge as presumptive HSCs. These nascent hematopoietic cells assume a rounded morphology, transiently occupy the subaortic space, and eventually enter the circulation via the caudal vein. Cell tracing showed that these cells subsequently populated the sites of definitive hematopoiesis (thymus and kidney), consistent with an HSC identity. HSC numbers depended on activity of the transcription factor Runx1, on blood flow, and on proper development of the dorsal aorta (features in common with mammals). This study captures the earliest events of the transition of endothelial cells to a hemogenic endothelium and demonstrates that embryonic hematopoietic progenitors directly differentiate from endothelial cells within a living organism.

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A Ras-like GTPase Is Involved in Hyphal Growth Guidance in the Filamentous Fungus Ashbya gossypii

Molecular Biology of the Cell ◽

10.1091/mbc.e04-02-0104 ◽

2004 ◽

Vol 15 (10) ◽

pp. 4622-4632 ◽

Cited By ~ 54

Author(s):

Yasmina Bauer ◽

Philipp Knechtle ◽

Jürgen Wendland ◽

Hanspeter Helfer ◽

Peter Philippsen

Keyword(s):

Fluorescent Protein ◽

Hyphal Growth ◽

Time Lapse ◽

Growth Direction ◽

Ashbya Gossypii ◽

Growth Guidance ◽

Characteristic Features ◽

Green Fluorescent ◽

Hyphal Tip

Characteristic features of morphogenesis in filamentous fungi are sustained polar growth at tips of hyphae and frequent initiation of novel growth sites (branches) along the extending hyphae. We have begun to study regulation of this process on the molecular level by using the model fungus Ashbya gossypii. We found that the A. gossypii Ras-like GTPase Rsr1p/Bud1p localizes to the tip region and that it is involved in apical polarization of the actin cytoskeleton, a determinant of growth direction. In the absence of RSR1/BUD1, hyphal growth was severely slowed down due to frequent phases of pausing of growth at the hyphal tip. During pausing events a hyphal tip marker, encoded by the polarisome component AgSPA2, disappeared from the tip as was shown by in vivo time-lapse fluorescence microscopy of green fluorescent protein-labeled AgSpa2p. Reoccurrence of AgSpa2p was required for the resumption of hyphal growth. In the Agrsr1/bud1Δ deletion mutant, resumption of growth occurred at the hyphal tip in a frequently uncoordinated manner to the previous axis of polarity. Additionally, hyphal filaments in the mutant developed aberrant branching sites by mislocalizing AgSpa2p thus distorting hyphal morphology. These results define AgRsr1p/Bud1p as a key regulator of hyphal growth guidance.

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The Positioning and Dynamics of Origins of Replication in the Budding Yeast Nucleus

The Journal of Cell Biology ◽

10.1083/jcb.152.2.385 ◽

2001 ◽

Vol 152 (2) ◽

pp. 385-400 ◽

Cited By ~ 115

Author(s):

Patrick Heun ◽

Thierry Laroche ◽

M.K. Raghuraman ◽

Susan M. Gasser

Keyword(s):

Nuclear Envelope ◽

Chromatin Structure ◽

Fluorescent Protein ◽

Time Lapse ◽

Yeast Cells ◽

G1 Phase ◽

Nuclear Pore ◽

Origin Firing ◽

Green Fluorescent

We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)–tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.

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Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages

Blood ◽

10.1182/blood.v96.2.719 ◽

2000 ◽

Vol 96 (2) ◽

pp. 719-726 ◽

Cited By ~ 397

Author(s):

Nicole Faust ◽

Florencio Varas ◽

Louise M. Kelly ◽

Susanne Heck ◽

Thomas Graf

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Time Lapse ◽

Neutrophil Granulocytes ◽

Egfp Gene ◽

Hematopoietic Stem ◽

Multipotent Progenitors ◽

Myelomonocytic Cells ◽

Green Fluorescent

Abstract Pluripotent hematopoietic stem cells have been studied extensively, but the events that occur during their differentiation remain largely uncharted. To develop a system that allows the differentiation of cultured multipotent progenitors by time-lapse fluorescence microscopy, myelomonocytic cells were labeled with green fluorescent protein (GFP) in vivo. This was achieved by knocking the enhanced GFP (EGFP) gene into the murine lysozyme M (lys) locus and using a targeting vector, which contains a neomycin resistant (neo) gene flanked by LoxP sites and “splinked” ends, to increase the frequency of homologous recombination. Analysis of the blood and bone marrow of thelys-EGFP mice revealed that most myelomonocytic cells, especially mature neutrophil granulocytes, were fluorescence-positive, while cells from other lineages were not. Removal of the neogene through breeding of the mice with the Cre-deleter strain led to an increased fluorescence intensity. Mice with an inactivation of both copies of the lys gene developed normally and were fertile.

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Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages

Blood ◽

10.1182/blood.v96.2.719.014k29_719_726 ◽

2000 ◽

Vol 96 (2) ◽

pp. 719-726 ◽

Cited By ~ 56

Author(s):

Nicole Faust ◽

Florencio Varas ◽

Louise M. Kelly ◽

Susanne Heck ◽

Thomas Graf

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Time Lapse ◽

Neutrophil Granulocytes ◽

Egfp Gene ◽

Hematopoietic Stem ◽

Multipotent Progenitors ◽

Myelomonocytic Cells ◽

Green Fluorescent

Pluripotent hematopoietic stem cells have been studied extensively, but the events that occur during their differentiation remain largely uncharted. To develop a system that allows the differentiation of cultured multipotent progenitors by time-lapse fluorescence microscopy, myelomonocytic cells were labeled with green fluorescent protein (GFP) in vivo. This was achieved by knocking the enhanced GFP (EGFP) gene into the murine lysozyme M (lys) locus and using a targeting vector, which contains a neomycin resistant (neo) gene flanked by LoxP sites and “splinked” ends, to increase the frequency of homologous recombination. Analysis of the blood and bone marrow of thelys-EGFP mice revealed that most myelomonocytic cells, especially mature neutrophil granulocytes, were fluorescence-positive, while cells from other lineages were not. Removal of the neogene through breeding of the mice with the Cre-deleter strain led to an increased fluorescence intensity. Mice with an inactivation of both copies of the lys gene developed normally and were fertile.

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Effect of MicroRNA-20a on Osteogenic Differentiation of Human Adipose Tissue-Derived Stem Cells

Cells Tissues Organs ◽

10.1159/000506304 ◽

2019 ◽

Vol 208 (3-4) ◽

pp. 148-157

Author(s):

Tao Luo ◽

Xueqin Yang ◽

Yan Sun ◽

Xinqi Huang ◽

Ling Zou ◽

...

Keyword(s):

Stem Cells ◽

Adipose Tissue ◽

Osteogenic Differentiation ◽

Fluorescent Protein ◽

Mrna Level ◽

Human Adipose Tissue ◽

Associated Proteins ◽

Green Fluorescent

Osteogenic differentiation of human adipose tissue-derived stem cells (hASCs) is a complex process that is regulated by multiple factors, including microRNAs (miRNAs). The miRNA miR-20a was shown to promote bone formation from bone marrow-derived mesenchymal stem cells. However, the role of miR-20a in osteogenic differentiation of hASCs remains unclear. In this study, we systematically evaluated the function of miR-20a in regulating hASC osteogenesis in vitro. hASCs were transduced with miR-20a-overexpressing and miR-20a-sponge lentiviral vectors, with green fluorescent protein (GFP) as a control. The results showed that miR-20a transcription was upregulated after hASC mineralization. Compared with the miR-20a-sponge, GFP, and hASC groups, the miR-20a-overexpressing group showed higher alkaline phosphatase (ALP) activity on days 7 and 14. Moreover, the mRNA level of ALP increased significantly in the miR-20a-overexpressing group on day 14. Furthermore, the protein of the target gene PPARγ was decreased, and the osteogenic differentiation-associated proteins ALP, osteocalcin, and RUNX2 were upregulated. hASCs anchored to HA/β-TCP revealed a healthy polygonal morphology and developed cytoplasmic extensions. miR-20a promoted osteogenic differentiation of the cell scaffold. Taken together, these data confirm that miRNA-20a promotes the osteogenesis of hASCs in vitro, and its essential role in vivo needs further investigation.

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Alf1p, a CLIP-170 Domain-containing Protein, Is Functionally and Physically Associated with α-Tubulin

The Journal of Cell Biology ◽

10.1083/jcb.144.1.113 ◽

1999 ◽

Vol 144 (1) ◽

pp. 113-124 ◽

Cited By ~ 38

Author(s):

Becket Feierbach ◽

Eva Nogales ◽

Kenneth H. Downing ◽

Tim Stearns

Keyword(s):

Fluorescent Protein ◽

Null Alleles ◽

Microtubule Associated Proteins ◽

Cytoplasmic Face ◽

Associated Proteins ◽

Green Fluorescent ◽

Β Tubulin

Tubulin is a heterodimer of α- and β-tubulin polypeptides. Assembly of the tubulin heterodimer in vitro requires the CCT chaperonin complex, and a set of five proteins referred to as the tubulin cofactors (Tian, F., Y. Huang, H. Rommelaere, J. Vandekerckhove, C. Ampe, and N.J. Cowan. 1996. Cell. 86:287–296; Tian, G., S.A. Lewis, B. Feierbach, T. Stearns, H. Rommelaere, C. Ampe, and N.J. Cowan. 1997. J. Cell Biol. 138:821–832). We report the characterization of Alf1p, the yeast ortholog of mammalian cofactor B. Alf1p interacts with α-tubulin in both two-hybrid and immunoprecipitation assays. Alf1p and cofactor B contain a single CLIP-170 domain, which is found in several microtubule-associated proteins. Mutation of the CLIP-170 domain in Alf1p disrupts the interaction with α-tubulin. Mutations in α-tubulin that disrupt the interaction with Alf1p map to a domain on the cytoplasmic face of α-tubulin; this domain is distinct from the region of interaction between α-tubulin and β-tubulin. Alf1p-green fluorescent protein (GFP) is able to associate with microtubules in vivo, and this localization is abolished either by mutation of the CLIP-170 domain in Alf1p, or by mutation of the Alf1p-binding domain in α-tubulin. Analysis of double mutants constructed between null alleles of ALF1 and PAC2, which encodes the other yeast α-tubulin cofactor, suggests that Alf1p and Pac2p act in the same pathway leading to functional α-tubulin. The phenotype of overexpression of ALF1 suggests that Alf1p can act to sequester α-tubulin from interaction with β-tubulin, raising the possibility that it plays a regulatory role in the formation of the tubulin heterodimer.

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BIM1Encodes a Microtubule-binding Protein in Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.8.12.2677 ◽

1997 ◽

Vol 8 (12) ◽

pp. 2677-2691 ◽

Cited By ~ 137

Author(s):

Katja Schwartz ◽

Kristy Richards ◽

David Botstein

Keyword(s):

Fluorescent Protein ◽

Synthetic Lethality ◽

Binding Protein ◽

Human Colon ◽

Wild Type ◽

Human Colon Cancer ◽

Microtubule Binding ◽

Green Fluorescent ◽

Two Hybrid

A previously uncharacterized yeast gene (YER016w) that we have named BIM1 (binding to microtubules) was obtained from a two-hybrid screen of a yeast cDNA library using as bait the entire coding sequence of TUB1 (encoding α-tubulin). Deletion of BIM1 results in a strong bilateral karyogamy defect, hypersensitivity to benomyl, and aberrant spindle behavior, all phenotypes associated with mutations affecting microtubules in yeast, and inviability at extreme temperatures (i.e., ≥37°C or ≤14°C). Overexpression of BIM1 in wild-type cells is lethal. A fusion of Bim1p with green fluorescent protein that complements the bim1Δ phenotypes allows visualization in vivo of both intranuclear spindles and extranuclear microtubules in otherwise wild-type cells. A bim1deletion displays synthetic lethality with deletion alleles ofbik1, num1, and bub3 as well as a limited subset of tub1 conditional-lethal alleles. A systematic study of 51 tub1 alleles suggests a correlation between specific failure to interact with Bim1p in the two-hybrid assay and synthetic lethality with thebim1Δ allele. The sequence of BIM1shows substantial similarity to sequences from organisms across the evolutionary spectrum. One of the human homologues, EB1, has been reported previously as binding APC, itself a microtubule-binding protein and the product of a gene implicated in the etiology of human colon cancer.

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Control of Microtubule Dynamics by Stu2p Is Essential for Spindle Orientation and Metaphase Chromosome Alignment in Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.12.9.2870 ◽

2001 ◽

Vol 12 (9) ◽

pp. 2870-2880 ◽

Cited By ~ 114

Author(s):

Karena A. Kosco ◽

Chad G. Pearson ◽

Paul S. Maddox ◽

Peijing Jeremy Wang ◽

Ian R. Adams ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Microtubule Dynamics ◽

Spindle Orientation ◽

Cytoplasmic Microtubule ◽

Chromosome Alignment ◽

In Vivo Analysis ◽

Cytoplasmic Microtubules ◽

Green Fluorescent

Stu2p is a member of a conserved family of microtubule-binding proteins and an essential protein in yeast. Here, we report the first in vivo analysis of microtubule dynamics in cells lacking a member of this protein family. For these studies, we have used a conditional Stu2p depletion strain expressing α-tubulin fused to green fluorescent protein. Depletion of Stu2p leads to fewer and less dynamic cytoplasmic microtubules in both G1 and preanaphase cells. The reduction in cytoplasmic microtubule dynamics is due primarily to decreases in both the catastrophe and rescue frequencies and an increase in the fraction of time microtubules spend pausing. These changes have significant consequences for the cell because they impede the ability of cytoplasmic microtubules to orient the spindle. In addition, recovery of fluorescence after photobleaching indicates that kinetochore microtubules are no longer dynamic in the absence of Stu2p. This deficiency is correlated with a failure to properly align chromosomes at metaphase. Overall, we provide evidence that Stu2p promotes the dynamics of microtubule plus-ends in vivo and that these dynamics are critical for microtubule interactions with kinetochores and cortical sites in the cytoplasm.

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