scholarly journals Specific association of an M-phase kinase with isolated mitotic spindles and identification of two of its substrates as MAP4 and MAP1B.

1991 ◽  
Vol 2 (11) ◽  
pp. 861-874 ◽  
Author(s):  
R M Tombes ◽  
J G Peloquin ◽  
G G Borisy
Keyword(s):  
Mitotic Cycle ◽  
Chinese Hamster ◽  
Mitotic Spindles ◽  
Microtubule Associated Proteins ◽  
M Phase ◽  
Associated Proteins ◽  
Specific Association ◽  
Cycle Dependent

Isolated mammalian (Chinese hamster ovary [CHO]) metaphase spindles were found to be enriched in a histone H1 kinase whose activity was mitotic-cycle dependent. Two substrates for the kinase were identified as MAP1B and MAP4. Partially purified spindle kinase retained activity for the spindle microtubule-associated proteins (MAPs) as well as brain and other tissue culture MAPs; on phosphorylation, spindle MAPs exhibited increased immunoreactivity with MPM-2, a monoclonal antibody specific for a subset of mitotic phosphoproteins. Immunofluorescence using an anti-thiophosphoprotein antibody localized in vitro phosphorylated spindle proteins to microtubule fibers, centrosomes, kinetochores, and midbodies. The fractionated spindle kinase was reactive with anti-human p34cdc2 antibodies and with an anti-human cyclin B but not an anti-human cyclin A antibody. We conclude that spindle MAPs undergo mitotic cycle-dependent phosphorylations in vivo and associate with a kinase that remains active on spindle isolation and may be related to p34cdc2.

scholarly journals Cell-cycle-dependent phosphorylation and activity of Chinese-hamster ovary topoisomerase II

1993 ◽  
Vol 293 (1) ◽  
pp. 297-304 ◽  
Author(s):  
D A Burden ◽  
L J Goldsmith ◽  
D M Sullivan
Keyword(s):  
Cell Cycle ◽  
Topoisomerase Ii ◽  
Chinese Hamster ◽  
Dna Topoisomerase ◽  
Protein Levels ◽  
Cleavage Activity ◽  
M Phase ◽  
Cycle Dependent

Cell-cycle-dependent protein levels and phosphorylation of DNA topoisomerase II in relation to its catalytic and cleavage activities were studied in Chinese-hamster ovary cells. Immunoreactive topoisomerase II protein levels were maximal in G2-phase cells, intermediate in S- and M-phase cells, and minimal in a predominantly G1-phase population. When the phosphorylation of topoisomerase II in vivo was corrected for differences in specific radioactivity of intracellular ATP, the apparent phosphorylation of S- and M-phase topoisomerase II was altered significantly. Relative phosphorylation in vivo was found to be greatest in M-phase cells and decreased in the other populations in the order: S > G2 > asynchronous. Phosphoserine was detected in every phase of the cell cycle, with a minor contribution of phosphothreonine demonstrated in M-phase cells. Topoisomerase II activity measured in vivo as 9-(4,6-O-ethylidene-beta-D-glucopyranosyl)-4′-demethylepipodophylloto xin (VP-16)-induced DNA double-strand breaks (determined by neutral filter elution) increased in the order: asynchronous < S < G2 < M. Topoisomerase II cleavage activity, assayed in vitro as the formation of covalent enzyme-DNA complexes, was lowest in S phase, intermediate in asynchronous and G2-phase cells, and maximal in M phase. Topoisomerase II decatenation activity was 1.6-1.8-fold greater in S-, G2- and M-phase populations relative to asynchronous cells. Therefore DNA topoisomerase II activity measured both in vivo and in vitro is maximal in M phase, that phase of the cell cycle with an intermediate level of immunoreactive topoisomerase II but the highest level of enzyme phosphorylation. The discordance between immunoreactive topoisomerase II protein levels, adjusted relative phosphorylation, catalytic activity, cleavage activity and amino acid residue(s) modified, suggests that the site of phosphorylation may be cell-cycle-dependent and critical in determining catalytic and cleavage activity.

scholarly journals Stu2p binds tubulin and undergoes an open-to-closed conformational change

2006 ◽  
Vol 172 (7) ◽  
pp. 1009-1022 ◽  
Author(s):  
Jawdat Al-Bassam ◽  
Mark van Breugel ◽  
Stephen C. Harrison ◽  
Anthony Hyman
Keyword(s):  
Conformational Change ◽  
Conformational Transition ◽  
Budding Yeast ◽  
Microtubule Dynamics ◽  
Open Structure ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Common

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

scholarly journals Marliolide Derivative Induces Melanosome Degradation via Nrf2/p62-Mediated Autophagy

2021 ◽  
Vol 22 (8) ◽  
pp. 3995
Author(s):  
Cheong-Yong Yun ◽  
Nahyun Choi ◽  
Jae Un Lee ◽  
Eun Jung Lee ◽  
Ji Young Kim ◽  
...  
Keyword(s):  
Related Factor ◽  
Melanin Pigmentation ◽  
Microtubule Associated Proteins ◽  
Melanocyte Stimulating Hormone ◽  
Associated Proteins ◽  
Autophagy Pathway ◽  
Nrf2 Activator ◽  
Promising Therapeutic Agent

Nuclear factor erythroid 2-related factor 2 (Nrf2), which is linked to autophagy regulation and melanogenesis regulation, is activated by marliolide. In this study, we investigated the effect of a marliolide derivative on melanosome degradation through the autophagy pathway. The effect of the marliolide derivative on melanosome degradation was investigated in α-melanocyte stimulating hormone (α-MSH)-treated melanocytes, melanosome-incorporated keratinocyte, and ultraviolet (UV)B-exposed HRM-2 mice (melanin-possessing hairless mice). The marliolide derivative, 5-methyl-3-tetradecylidene-dihydro-furan-2-one (DMF02), decreased melanin pigmentation by melanosome degradation in α-MSH-treated melanocytes and melanosome-incorporated keratinocytes, evidenced by premelanosome protein (PMEL) expression, but did not affect melanogenesis-associated proteins. The UVB-induced hyperpigmentation in HRM-2 mice was also reduced by a topical application of DMF02. DMF02 activated Nrf2 and induced autophagy in vivo, evidenced by decreased PMEL in microtubule-associated proteins 1A/1B light chain 3B (LC3)-II-expressed areas. DMF02 also induced melanosome degradation via autophagy in vitro, and DMF02-induced melanosome degradation was recovered by chloroquine (CQ), which is a lysosomal inhibitor. In addition, Nrf2 silencing by siRNA attenuated the DMF02-induced melanosome degradation via the suppression of p62. DMF02 induced melanosome degradation in melanocytes and keratinocytes by regulating autophagy via Nrf2-p62 activation. Therefore, Nrf2 activator could be a promising therapeutic agent for reducing hyperpigmentation.

scholarly journals Stable expression of heterologous microtubule-associated proteins (MAPs) in Chinese hamster ovary cells: evidence for differing roles of MAPs in microtubule organization.

1994 ◽  
Vol 126 (4) ◽  
pp. 1017-1029 ◽  
Author(s):  
S Barlow ◽  
M L Gonzalez-Garay ◽  
R R West ◽  
J B Olmsted ◽  
F Cabral
Keyword(s):  
Sodium Butyrate ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Constitutive Expression ◽  
Microtubule Assembly ◽  
Western Blots ◽  
Transfected Cells ◽  
Microtubule Associated Proteins ◽  
Associated Proteins

To study the effects of microtubule-associated proteins (MAPs) on in vivo microtubule assembly, cDNAs containing the complete coding sequences of a Drosophila 205-kD heat stable MAP, human MAP 4, and human tau were stably transfected into CHO cells. Constitutive expression of the transfected genes was low in most cases and had no obvious effects on the viability of the transfected cell lines. High levels of expression, as judged by Western blots, immunofluorescence, and Northern blots, could be induced by treating cells with sodium butyrate. High levels of MAPs were maintained for at least 24-48 h after removal of the sodium butyrate. Immunofluorescence analysis indicated that all three MAPs bound to cellular microtubules, but only the transfected tau caused a rearrangement of microtubules into bundles. Despite high levels of expression of these exogenous MAPs and the bundling of microtubules in cells expressing tau, transfected cells had normal levels of assembled and unassembled tubulin. With the exception of the tau-induced bundles, microtubules in transfected cells showed the same sensitivity as control cells to microtubule depolymerization by Colcemid. Further, all three MAPs were ineffective in reversing the taxol-dependent phenotype of a CHO mutant cell line. The absence of a quantitative effect of any of these heterologous proteins on the assembly of tubulin suggests that these MAPs may have different roles in vivo from those inferred previously from in vitro experiments.

scholarly journals Yeast proteins associated with microtubules in vitro and in vivo.

1992 ◽  
Vol 3 (1) ◽  
pp. 29-47 ◽  
Author(s):  
G Barnes ◽  
K A Louie ◽  
D Botstein
Keyword(s):  
Self Assembly ◽  
Synthetic Peptides ◽  
Immunofluorescence Microscopy ◽  
The Self ◽  
Microtubule Associated Proteins ◽  
Amino Terminal ◽  
Novel Proteins ◽  
Associated Proteins

Conditions were established for the self-assembly of milligram amounts of purified Saccharomyces cerevisiae tubulin. Microtubules assembled with pure yeast tubulin were not stabilized by taxol; hybrid microtubules containing substoichiometric amounts of bovine tubulin were stabilized. Yeast microtubule-associated proteins (MAPs) were identified on affinity matrices made from hybrid and all-bovine microtubules. About 25 yeast MAPs were isolated. The amino-terminal sequences of several of these were determined: three were known metabolic enzymes, two were GTP-binding proteins (including the product of the SAR1 gene), and three were novel proteins not found in sequence databases. Affinity-purified antisera were generated against synthetic peptides corresponding to two of the apparently novel proteins (38 and 50 kDa). Immunofluorescence microscopy showed that both these proteins colocalize with intra- and extranuclear microtubules in vivo.

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

1999 ◽  
Vol 144 (1) ◽  
pp. 113-124 ◽  
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.

scholarly journals A microtubule-associated protein antigen unique to mitotic spindle microtubules in PtK1 cells.

1983 ◽  
Vol 96 (2) ◽  
pp. 424-434 ◽  
Author(s):  
J G Izant ◽  
J A Weatherbee ◽  
J R McIntosh
Keyword(s):  
Mitotic Spindle ◽  
Microtubule Network ◽  
Mitotic Apparatus ◽  
Microtubule Associated Proteins ◽  
Immunoglobulin Preparations ◽  
Rapid Dissolution ◽  
Associated Proteins ◽  
Cultured Human Cells

Microtubule-associated proteins (MAPs) that copurify with tubulin through multiple cycles of in vitro assembly have been implicated as regulatory factors and effectors in the in vivo activity of microtubules. As an approach to the analysis of the functions of these molecules, a collection of lymphocyte hybridoma monoclonal antibodies has been generated using MAPs from HeLa cell microtubule protein as antigen. Two of the hybridoma clones secrete IgGs that bind to distinct sites on what appears to be a 200,000-dalton polypeptide. Both immunoglobulin preparations stain interphase and mitotic apparatus microtubules in cultured human cells. One of the clones (N-3B4.3.10) secretes antibody that reacts only with cells of human origin, while antibody from the other hybridoma (N-2B5.11.2) cross-reacts with BSC and PtK1 cells, but not with 3T3 cells. In PtK1 cells the N-2B5 antigen is associated with the microtubules of the mitotic apparatus, but there is no staining of the interphase microtubule array; rather, the antibody stains an ill-defined juxtanuclear structure. Further, neither antibody stains vinblastine crystals in either human or marsupial cells at any stage of the cell cycle. N-2B5 antibody microinjected into living PtK1 cells binds to the mitotic spindle, but does not cause a rapid dissolution of either mitotic or interphase microtubule structures. When injected before the onset of anaphase, however, the N-2B5 antibody inhibits proper chromosome partition in mitotic PtK1 cells. N-2B5 antibody injected into interphase cells causes a redistribution of MAP antigen onto the microtubule network.

scholarly journals Neuroblastoma therapy: what is in the pipeline?

2011 ◽  
Vol 18 (6) ◽  
pp. R213-R231 ◽  
Author(s):  
Carla S Verissimo ◽  
Jan J Molenaar ◽  
Carlos P Fitzsimons ◽  
Erno Vreugdenhil
Keyword(s):  
Cathepsin L ◽  
Molecular Targets ◽  
Kinase Gene ◽  
Microtubule Associated Proteins ◽  
Micro Rnas ◽  
Associated Proteins ◽  
Selection Of

Despite the expansion of knowledge about neuroblastoma (NB) in recent years, the therapeutic outcome for children with a high-risk NB has not significantly improved. Therefore, more effective therapies are needed. This might be achieved by aiming future efforts at recently proposed but not yet developed targets for NB therapy. In this review, we discuss the recently proposed molecular targets that are in clinical trials and, in particular, those that are not yet explored in the clinic. We focus on the selection of these molecular targets for which promisingin vitroandin vivoresults have been obtained by silencing/inhibiting them. In addition, these selected targets are involved at least in one of the NB tumorigenic processes: proliferation, anti-apoptosis, angiogenesis and/or metastasis. In particular, we will review a recently proposed target, the microtubule-associated proteins (MAPs) encoded by doublecortin-like kinase gene (DCLK1).DCLK1-derived MAPs are crucial for proliferation and survival of neuroblasts and are highly expressed not only in NB but also in other tumours such as gliomas. Additionally, we will discuss neuropeptide Y, its Y2 receptor and cathepsin L as examples of targets to decrease angiogenesis and metastasis of NB. Furthermore, we will review the micro-RNAs that have been proposed as therapeutic targets for NB. Detailed investigation of these not yet developed targets as well as exploration of multi-target approaches might be the key to a more effective NB therapy, i.e. increasing specificity, reducing toxicity and avoiding long-term side effects.

scholarly journals Microtubule associated proteins and motors required for ectopic microtubule array formation in S. cerevisiae

2021 ◽  
Author(s):  
Brianna R. King ◽  
Janet B. Meehl ◽  
Tamira Vojnar ◽  
Mark Winey ◽  
Eric G. Muller ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Polymerase Activity ◽  
Nucleation Site ◽  
Microtubule Nucleation ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
A Subunit ◽  
Nucleation Activity

AbstractThe mitotic spindle is resilient to perturbation due to the concerted, and sometimes redundant, action of motors and microtubule-associated proteins. Here we utilize an inducible ectopic microtubule nucleation site in the nucleus of Saccharomyces cerevisiae to study three necessary steps in the formation of a bipolar array: the recruitment of the γ-tubulin complex, nucleation and elongation of microtubules, and the organization of microtubules relative to each other. This novel tool, an Spc110 chimera, reveals previously unreported roles of the microtubule-associated proteins Stu2, Bim1, and Bik1, and the motors Vik1 and Kip3. We report that Stu2 and Bim1 are required for nucleation and that Bik1 and Kip3 promote nucleation at the ectopic site. Stu2, Bim1, and Kip3 join their homologs XMAP215, EB1 and kinesin-8 as promoters of microtubule nucleation, while Bik1 promotes MT nucleation indirectly via its role in SPB positioning. Further, we find that the nucleation activity of Stu2 in vivo correlates with its polymerase activity in vitro. Finally, we provide the first evidence that Vik1, a subunit of Kar3/Vik1 kinesin-14, promotes microtubule minus end focusing at the ectopic site.

scholarly journals The yeast homologue of the microtubule-associated protein Lis1 interacts with the sumoylation machinery and a SUMO-targeted ubiquitin ligase

2012 ◽  
Vol 23 (23) ◽  
pp. 4552-4566 ◽  
Author(s):  
Annabel Alonso ◽  
Sonia D'Silva ◽  
Maliha Rahman ◽  
Pam B. Meluh ◽  
Jacob Keeling ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Ubiquitin Ligases ◽  
Microtubule Associated Proteins ◽  
Sumo Protease ◽  
Cellular Processes ◽  
Associated Proteins ◽  
Novel Model ◽  
Intracellular Motility

Microtubules and microtubule-associated proteins are fundamental for multiple cellular processes, including mitosis and intracellular motility, but the factors that control microtubule-associated proteins (MAPs) are poorly understood. Here we show that two MAPs—the CLIP-170 homologue Bik1p and the Lis1 homologue Pac1p—interact with several proteins in the sumoylation pathway. Bik1p and Pac1p interact with Smt3p, the yeast SUMO; Ubc9p, an E2; and Nfi1p, an E3. Bik1p interacts directly with SUMO in vitro, and overexpression of Smt3p and Bik1p results in its in vivo sumoylation. Modified Pac1p is observed when the SUMO protease Ulp1p is inactivated. Both ubiquitin and Smt3p copurify with Pac1p. In contrast to ubiquitination, sumoylation does not directly tag the substrate for degradation. However, SUMO-targeted ubiquitin ligases (STUbLs) can recognize a sumoylated substrate and promote its degradation via ubiquitination and the proteasome. Both Pac1p and Bik1p interact with the STUbL Nis1p-Ris1p and the protease Wss1p. Strains deleted for RIS1 or WSS1 accumulate Pac1p conjugates. This suggests a novel model in which the abundance of these MAPs may be regulated via STUbLs. Pac1p modification is also altered by Kar9p and the dynein regulator She1p. This work has implications for the regulation of dynein's interaction with various cargoes, including its off-loading to the cortex.

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