Evidence that estramustine binds MAP-1A to inhibit type IV collagenase secretion

1991 ◽  
Vol 98 (1) ◽  
pp. 55-63
Author(s):  
M.E. Stearns ◽  
M. Wang ◽  
O. Sousa
Keyword(s):  
Polyclonal Antibodies ◽  
Drug Uptake ◽  
Type Iv Collagenase ◽  
Quantitative Evidence ◽  
Microtubule Associated Proteins ◽  
Type Iv ◽  
Associated Proteins ◽  
Uptake Studies ◽  
Microtubule Networks

Estramustine is a novel anti-microtubule drug shown to bind MAP-1 and MAP-2 (microtubule-associated proteins) in vitro. In this paper we have shown that estramustine specifically binds MAP-1A in Du 145a cells, resulting in disruption of MAP-1A microtubules and inhibition of type IV collagenase secretion. Immunofluorescence studies revealed that at 30 microM levels estramustine blocked type IV collagenase secretion by partial disruption of the MAP-1A microtubule networks. Immunoprecipitation studies with polyclonal antibodies provided quantitative evidence that 30–60 microM estramustine blocked secretion of a 105 × 10(3) Mr type IV collagenase. Pulse-labeling experiments confirmed that the effect was not a result of inhibition of either protein synthesis or altered rates of type IV collagenase turnover. Finally, drug uptake studies with [3H]estramustine, scintillation counting and fluorography demonstrated that the principal target of the drug was MAP-1A. For the first time we have shown that the drug blocks secretion by binding MAP-1A and causing incomplete disruption of the microtubule networks.

A protein related to brain microtubule-associated protein MAP1B is a component of the mammalian centrosome

1994 ◽  
Vol 107 (2) ◽  
pp. 601-611 ◽  
Author(s):  
J.E. Dominguez ◽  
B. Buendia ◽  
C. Lopez-Otin ◽  
C. Antony ◽  
E. Karsenti ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Polyclonal Antibodies ◽  
Microtubule Organizing Center ◽  
Microtubule Associated Proteins ◽  
Organizing Center ◽  
Associated Proteins ◽  
Pericentriolar Material ◽  
Peptide Maps

The centrosome is the main microtubule organizing center of mammalian cells. Structurally, it is composed of a pair of centrioles surrounded by a fibro-granular material (the pericentriolar material) from which microtubules are nucleated. However, the nature of centrosomal molecules involved in microtubules nucleation is still obscure. Since brain microtubule-associated proteins (MAPs) lower the critical tubulin concentration required for microtubule nucleation in tubulin solution in vitro, we have examined their possible association with centrosomes. By immunofluorescence, monoclonal and polyclonal antibodies raised against MAP1B stain the centrosome in cultured cells as well as purified centrosomes, whereas antibodies raised against MAP2 give a completely negative reaction. The MAP1B-related antigen is localized to the pericentriolar material as revealed by immunoelectron microscopy. In preparations of purified centrosomes analyzed on poly-acrylamide gels, a protein that migrates as brain MAP1B is present. After blotting on nitrocellulose, it is decorated by anti-MAP1B antibodies and the amino acid sequence of proteolytic fragments of this protein is similar to brain MAP1B. Moreover, brain MAP1B and its centrosomal counterpart share the same phosphorylation features and have similar peptide maps. These data strongly suggest that a protein homologue to MAP1B is present in centrosomes and it is a good candidate for being involved in the nucleating activity of the pericentriolar material.

scholarly journals Plant and mouse EB1 proteins have opposite intrinsic properties on the dynamic instability of microtubules.

2020 ◽  
Author(s):  
Arthur T Molines ◽  
Virginie Stoppin-Mellet ◽  
Isabelle Arnal ◽  
Frédéric M Coquelle
Keyword(s):  
Dynamic Instability ◽  
Cellular Organization ◽  
Cell Type ◽  
Intrinsic Properties ◽  
Microtubule Organization ◽  
Vitro Assay ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Microtubule Networks

Abstract Objective Most eukaryotic cells contain microtubule filaments, which play central roles in intra-cellular organization. However, microtubule networks have a wide variety of architectures from one cell type and organism to another. Nonetheless, the sequences of tubulins, of Microtubule Associated proteins (MAPs) and the structure of microtubules are usually well conserved throughout the evolution. MAPs being known to be responsible for regulating microtubule organization and dynamics, this raises the question of the conservation of their intrinsic properties. Indeed, knowing how the intrinsic properties of individual MAPs differ between organisms might enlighten our understanding of how distinct microtubule networks are built. End-Binding protein 1 (EB1), first described as a MAP in yeast, is conserved in plants and mammals. The intrinsic properties of the mammalian and the yeast EB1 proteins have been well described in the literature but, to our knowledge, the intrinsic properties of EB1 from plant and mammals have not been compared thus far.Results Here, using an in vitro assay, we discovered that plant and mammalian EB1 purified proteins have different intrinsic properties on microtubule dynamics. Indeed, the mammalian EB1 protein increases microtubules dynamic while the plant EB1 protein stabilizes them.

Association of p34cdc2/cyclin B complex with microtubules in starfish oocytes

1993 ◽  
Vol 105 (4) ◽  
pp. 873-881 ◽  
Author(s):  
K. Ookata ◽  
S. Hisanaga ◽  
E. Okumura ◽  
T. Kishimoto
Keyword(s):  
Complex Form ◽  
Cyclin B ◽  
Microtubule Associated Proteins ◽  
Inactive Form ◽  
Microtubular Cytoskeleton ◽  
Associated Proteins ◽  
Meiotic Spindles ◽  
Microtubule Networks ◽  
Brain Microtubules

The microtubular cytoskeleton exhibits a dramatic reorganization, progressing from interphase radial arrays to a mitotic spindle at the G2/M transition. Although this reorganization has been suspected to be caused by maturation promoting factor (MPF: p34cdc2/cyclin B complex), little is known about how p34cdc2 kinase controls microtubule networks. We provide evidence of the direct association of the p34cdc2/cyclin B complex with microtubules in starfish oocytes. Anti-cyclin B staining of detergent-treated oocytes, isolated asters and meiotic spindles revealed fluorescence associated with microtubule fibers, chromosomes and centrosomes. Microtubules prepared from starfish oocytes were associated with cyclin B and p34cdc2 proteins. Microtubule-bound p34cdc2 and cyclin B were released from microtubules by a high-salt solution and possessed a complex form as shown by the adsorption to suc1-beads and by immunoprecipitation with the anti-cyclin B antibody. The p34cdc2/cyclin B complex associated to microtubules had high histone H1 kinase activity at meiotic metaphase. However, it was not necessary for the p34cdc2/cyclin B complex to be active for microtubule binding, as an inactive form in immature oocytes was also observed to bind to microtubules. The coprecipitation of suc1-column purified p34cdc2/cyclin B with purified porcine brain microtubules in the presence of starfish oocyte microtubule-associated proteins (MAPs) indicates that the association of p34cdc2/cyclin B with microtubules in vitro is mediated by MAPs.

scholarly journals Plant and mouse EB1 proteins have opposite intrinsic properties on the dynamic instability of microtubules.

2020 ◽  
Author(s):  
Arthur T Molines ◽  
Virginie Stoppin-Mellet ◽  
Isabelle Arnal ◽  
Frédéric M Coquelle
Keyword(s):  
Dynamic Instability ◽  
Eukaryotic Cells ◽  
Cellular Organization ◽  
Cell Type ◽  
Intrinsic Properties ◽  
Microtubule Organization ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Microtubule Networks

Abstract Objective Most eukaryotic cells contain microtubule filaments, which play central roles in intra-cellular organization. However, microtubule networks have a wide variety of architectures from one cell type and organism to another. Nonetheless, the sequences of tubulins, of Microtubule Associated proteins (MAPs) and the structure of microtubules are usually well conserved throughout the evolution. MAPs being known to be responsible for regulating microtubule organization and dynamics, this raises the question of the conservation of their intrinsic properties. Indeed, knowing how the intrinsic properties of individual MAPs differ between organisms might enlighten our understanding of how distinct microtubule networks are built. End-Binding protein 1 (EB1), first described as a MAP in yeast, is conserved in plants and mammals. The intrinsic properties of the mammalian and the yeast EB1 proteins have been well described in the literature but, to our knowledge, the intrinsic properties of EB1 from plant and mammals have not been compared thus far.Results Here, using an in vitro assay, we discovered that plant and mammalian EB1 purified proteins have different intrinsic properties on microtubule dynamics. Indeed, the mammalian EB1 protein increases microtubules dynamic while the plant EB1 protein stabilizes them.

Analysis of the Mechanism of Microtubule Dynamic Instability Using a UV Microbeam to Sever Elongating Microtubules

1988 ◽  
Vol 46 ◽  
pp. 122-123
Author(s):  
R.A Walker ◽  
S. Inoue ◽  
E.D. Salmon
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic ◽  
Gtp Hydrolysis ◽  
Abrupt Transition ◽  
Microtubule Associated Proteins ◽  
Asymmetrical Structure ◽  
Associated Proteins

Microtubules polymerized in vitro from tubulin purified free of microtubule-associated proteins exhibit dynamic instability (1,2,3). Free microtubule ends exist in persistent phases of elongation or rapid shortening with infrequent, but, abrupt transitions between these phases. The abrupt transition from elongation to rapid shortening is termed catastrophe and the abrupt transition from rapid shortening to elongation is termed rescue. A microtubule is an asymmetrical structure. The plus end grows faster than the minus end. The frequency of catastrophe of the plus end is somewhat greater than the minus end, while the frequency of rescue of the plus end in much lower than for the minus end (4).The mechanism of catastrophe is controversial, but for both the plus and minus microtubule ends, catastrophe is thought to be dependent on GTP hydrolysis. Microtubule elongation occurs by the association of tubulin-GTP subunits to the growing end. Sometime after incorporation into an elongating microtubule end, the GTP is hydrolyzed to GDP, yielding a core of tubulin-GDP capped by tubulin-GTP (“GTP-cap”).

Molecular architecture and functions of the neuronal cytoskeleton

1990 ◽  
Vol 48 (3) ◽  
pp. 2-3
Author(s):  
Nobutaka Hirokawa
Keyword(s):  
Major Elements ◽  
Molecular Structures ◽  
Organelle Transport ◽  
Molecular Architecture ◽  
Neuronal Morphogenesis ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
And Function ◽  
Small Clusters

In this symposium I will present our studies about the molecular architecture and function of the cytomatrix of the nerve cells. The nerve cell is a highly polarized cell composed of highly branched dendrites, cell body, and a single long axon along the direction of the impulse propagation. Each part of the neuron takes characteristic shapes for which the cytoskeleton provides the framework. The neuronal cytoskeletons play important roles on neuronal morphogenesis, organelle transport and the synaptic transmission. In the axon neurofilaments (NF) form dense arrays, while microtubules (MT) are arranged as small clusters among the NFs. On the other hand, MTs are distributed uniformly, whereas NFs tend to run solitarily or form small fascicles in the dendrites Quick freeze deep etch electron microscopy revealed various kinds of strands among MTs, NFs and membranous organelles (MO). These structures form major elements of the cytomatrix in the neuron. To investigate molecular nature and function of these filaments first we studied molecular structures of microtubule associated proteins (MAP1A, MAP1B, MAP2, MAP2C and tau), and microtubules reconstituted from MAPs and tubulin in vitro. These MAPs were all fibrous molecules with different length and formed arm like projections from the microtubule surface.

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 Microtubules and microtubule-associated proteins from the nematode Caenorhabditis elegans: periodic cross-links connect microtubules in vitro.

1986 ◽  
Vol 103 (1) ◽  
pp. 23-31 ◽  
Author(s):  
E J Aamodt ◽  
J G Culotti
Keyword(s):  
Caenorhabditis Elegans ◽  
Apparent Molecular Weight ◽  
Cross Link ◽  
Nematode Caenorhabditis Elegans ◽  
Microtubule Associated Proteins ◽  
C Elegans ◽  
Associated Proteins ◽  
Cross Links ◽  
The Cross

The nematode Caenorhabditis elegans should be an excellent model system in which to study the role of microtubules in mitosis, embryogenesis, morphogenesis, and nerve function. It may be studied by the use of biochemical, genetic, molecular biological, and cell biological approaches. We have purified microtubules and microtubule-associated proteins (MAPs) from C. elegans by the use of the anti-tumor drug taxol (Vallee, R. B., 1982, J. Cell Biol., 92:435-44). Approximately 0.2 mg of microtubules and 0.03 mg of MAPs were isolated from each gram of C. elegans. The C. elegans microtubules were smaller in diameter than bovine microtubules assembled in vitro in the same buffer. They contained primarily 9-11 protofilaments, while the bovine microtubules contained 13 protofilaments. The principal MAP had an apparent molecular weight of 32,000 and the minor MAPs were 30,000, 45,000, 47,000, 50,000, 57,000, and 100,000-110,000 mol wt as determined by SDS-gel electrophoresis. The microtubules were observed, by electron microscopy of negatively stained preparations, to be connected by stretches of highly periodic cross-links. The cross-links connected the adjacent protofilaments of aligned microtubules, and occurred at a frequency of one cross-link every 7.7 +/- 0.9 nm, or one cross-link per tubulin dimer along the protofilament. The cross-links were removed when the MAPs were extracted from the microtubules with 0.4 M NaCl. The cross-links then re-formed when the microtubules and the MAPs were recombined in a low salt buffer. These results strongly suggest that the cross-links are composed of MAPs.

scholarly journals Identity and Origin of the ATPase activity associated with neuronal microtubules. I. The ATPase activity is associated with membrane vesicles.

1983 ◽  
Vol 96 (5) ◽  
pp. 1298-1305 ◽  
Author(s):  
D B Murphy ◽  
R R Hiebsch ◽  
K T Wallis
Keyword(s):  
Molecular Weight ◽  
Atpase Activity ◽  
High Molecular Weight ◽  
Brain Tissue ◽  
Membrane Vesicles ◽  
Microtubule Associated Proteins ◽  
In Vitro Assembly ◽  
Microtubule Protein ◽  
Associated Proteins

Microtubule protein purified from brain tissue by cycles of in vitro assembly-disassembly contains ATPase activity that has been postulated to be associated with microtubule-associated proteins (MAPs) and therefore significant for studies of microtubule-dependent motility. In this paper we demonstrate that greater than 90% of the ATPase activity is particulate in nature and may be derived from contaminating membrane vesicles. We also show that the MAPs (MAP-1, MAP-2, and tau factors) and other high molecular weight polypeptides do not contain significant amounts of ATPase activity. These findings do not support the concept of "brain dynein" or of MAPs with ATPase activity.

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