scholarly journals A polymer-dependent increase in phosphorylation of beta-tubulin accompanies differentiation of a mouse neuroblastoma cell line.

1985 ◽  
Vol 100 (3) ◽  
pp. 764-774 ◽  
Author(s):  
D L Gard ◽  
M W Kirschner
Keyword(s):  
Neurite Outgrowth ◽  
Isoelectric Focusing ◽  
Developmental Regulation ◽  
Neuroblastoma Cell ◽  
Microtubule Assembly ◽  
Beta Tubulin ◽  
Fourfold Increase ◽  
Cell Extracts ◽  
Mouse Neuroblastoma ◽  
Sds Page

We have examined the phosphorylation of cellular microtubule proteins during differentiation and neurite outgrowth in N115 mouse neuroblastoma cells. N115 differentiation, induced by serum withdrawal, is accompanied by a fourfold increase in phosphorylation of a 54,000-mol-wt protein identified as a specific isoform of beta-tubulin by SDS PAGE, two-dimensional isoelectric focusing/SDS PAGE, and immunoprecipitation with a specific monoclonal antiserum. Isoelectric focusing/SDS PAGE of [35S]methionine-labeled cell extracts revealed that the phosphorylated isoform of beta-tubulin, termed beta 2, is one of three isoforms detected in differentiated N115 cells, and is diminished in amounts in the undifferentiated cells. Taxol, a drug which promotes microtubule assembly, stimulates phosphorylation of beta-tubulin in both differentiated and undifferentiated N115 cells. In contrast, treatment of differentiated cells with either colcemid or nocodazole causes a rapid decrease in beta-tubulin phosphorylation. Thus, the phosphorylation of beta-tubulin in N115 cells is coupled to the levels of cellular microtubules. The observed increase in beta-tubulin phosphorylation during differentiation then reflects developmental regulation of microtubule assembly during neurite outgrowth, rather than developmental regulation of a tubulin kinase activity.

Adriamycin promotes neurite outgrowth in the “neurite-minus” N1A-103 mouse neuroblastoma cell line

1992 ◽  
Vol 203 (1) ◽  
pp. 72-79 ◽  
Author(s):  
Jean Christophe Larcher ◽  
Laurence Cordeau-Lossouarn ◽  
Georges Romey ◽  
François Gros ◽  
Bernard Croizat ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Cell Line ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cell Line ◽  
Mouse Neuroblastoma Cell ◽  
Mouse Neuroblastoma

scholarly journals Estramustine binds a MAP-1-like protein to inhibit microtubule assembly in vitro and disrupt microtubule organization in DU 145 cells.

1988 ◽  
Vol 107 (6) ◽  
pp. 2647-2656 ◽  
Author(s):  
M E Stearns ◽  
M Wang ◽  
K D Tew ◽  
L I Binder
Keyword(s):  
Tumor Cells ◽  
Microtubule Assembly ◽  
Microtubule Organization ◽  
Electron Microscopic ◽  
Beta Tubulin ◽  
Western Blots ◽  
Sds Page ◽  
Microscopic Studies

The twofold purpose of the study was (a) to determine if a MAP-1-like protein was expressed in human prostatic DU 145 cells and (b) to demonstrate whether a novel antimicrotubule drug, estramustine, binds the MAP-1-like protein to disrupt microtubules. SDS-PAGE and Western blots showed that a 330-kD protein was associated with microtubules isolated in an assembly buffer containing 10 microM taxol and 10 mM adenylylimidodiphosphate. After purification to homogeneity on an A5m agarose column, the 330-kD protein was found to promote 6 S tubulin assembly. Turbidimetric (A350), SDS-PAGE, and electron microscopic studies revealed that micromolar estramustine inhibited assembly promoted by the 330-kD protein. Similarly, estramustine inhibited binding of the 330-kD protein to 6-S microtubules independently stimulated to assemble with taxol. Immunofluorescent studies with beta-tubulin antibody (27B) and MAP-1 antibody (MI-AI) revealed that 60 microM estramustine (a) caused disassembly of MAP-1 microtubules in DU 145 cells and (b) removed MAP-1 from the surfaces of microtubules stabilized with 0.1 microM taxol. Taken together the data suggested that estramustine binds to a 330-kD MAP-1-like protein to disrupt microtubules in tumor cells.

β-Casomorphin-5 stimulates neurite outgrowth in a mouse neuroblastoma cell line (Neuro-2a)

1998 ◽  
Vol 251 (2) ◽  
pp. 97-100 ◽  
Author(s):  
Minoru Sakaguchi ◽  
Kouichi Murayama ◽  
Kozue Yabe ◽  
Motonobu Satoh ◽  
Masao Takeuchi ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Cell Line ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cell Line ◽  
Mouse Neuroblastoma Cell ◽  
Mouse Neuroblastoma

Spastin Interacts with CRMP2 to Regulate Neurite Outgrowth by Controlling Microtubule Dynamics through Phosphorylation Modifications

2020 ◽  
Vol 19 ◽  
Author(s):  
Sumei Li ◽  
Jifeng Zhang ◽  
Jiaqi Zhang ◽  
Jiong Li ◽  
Longfei Cheng ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Hippocampal Neurons ◽  
Neuronal Development ◽  
Phosphorylation Site ◽  
Microtubule Dynamics ◽  
Microtubule Assembly ◽  
C Terminus ◽  
Mediator Protein ◽  
Branch Formation

Aims: Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal development, and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve injury. Background: Microtubule polymerization and severing are the basis for the neurite outgrowth and branch formation. Collapsin response mediator protein 2 (CRMP2) regulates axonal growth and branching as a binding partner of the tubulin heterodimer to promote microtubule assembly. And spastin participates in the growth and regeneration of neurites by severing microtubules into small segments. However, how CRMP2 and spastin cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated. Objective: To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin. Method: Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites. Result: We first demonstrated that CRMP2 interacted with spastin to promote the neurite outgrowth and branch formation. Furthermore, our results identified that phosphorylation modification failed to alter the binding affinities of CRMP2 for spastin, but inhibited their binding to microtubules. CRMP2 interacted with the MTBD domain of spastin via its C-terminus, and blocking the binding sites of them inhibited the outgrowth and branch formation of neurites. In addition, we confirmed one phosphorylation site S210 at spastin in hippocampal neurons and phosphorylation spastin at site S210 promoted the neurite outgrowth but not branch formation by remodeling microtubules. Conclusion: Taken together, our data demonstrated that the interaction of CRMP2 and spastin is required for neurite outgrowth and branch formation and their interaction is not regulated by their phosphorylation.

scholarly journals Increased microtubule assembly in bovine brain tubulin lacking the type III isotype of beta-tubulin.

1990 ◽  
Vol 265 (3) ◽  
pp. 1794-1799
Author(s):  
A Banerjee ◽  
M C Roach ◽  
P Trcka ◽  
R F Ludueña
Keyword(s):  
Bovine Brain ◽  
Microtubule Assembly ◽  
Beta Tubulin ◽  
Type Iii ◽  
Brain Tubulin

scholarly journals Structural analysis of mutations in the Drosophila beta 2-tubulin isoform reveals regions in the beta-tubulin molecular required for general and for tissue-specific microtubule functions.

Genetics ◽  
1995 ◽  
Vol 139 (1) ◽  
pp. 267-286 ◽  
Author(s):  
J D Fackenthal ◽  
J A Hutchens ◽  
F R Turner ◽  
E C Raff
Keyword(s):  
Amino Acid ◽  
Three Dimensional ◽  
Variable Region ◽  
Internal Variable ◽  
Microtubule Assembly ◽  
Dimensional Structure ◽  
Wild Type ◽  
Beta Tubulin ◽  
Assembly Kinetics ◽  
Beta 2

Abstract We have determined the lesions in a number of mutant alleles of beta Tub85D, the gene that encodes the testis-specific beta 2-tubulin isoform in Drosophila melanogaster. Mutations responsible for different classes of functional phenotypes are distributed throughout the beta 2-tubulin molecule. There is a telling correlation between the degree of phylogenetic conservation of the altered residues and the number of different microtubule categories disrupted by the lesions. The majority of lesions occur at positions that are evolutionarily highly conserved in all beta-tubulins; these lesions disrupt general functions common to multiple classes of microtubules. However, a single allele B2t6 contains an amino acid substitution within an internal cluster of variable amino acids that has been identified as an isotype-defining domain in vertebrate beta-tubulins. Correspondingly, B2t6 disrupts only a subset of microtubule functions, resulting in misspecification of the morphology of the doublet microtubules of the sperm tail axoneme. We previously demonstrated that beta 3, a developmentally regulated Drosophila beta-tubulin isoform, confers the same restricted morphological phenotype in a dominant way when it is coexpressed in the testis with wild-type beta 2-tubulin. We show here by complementation analysis that beta 3 and the B2t6 product disrupt a common aspect of microtubule assembly. We therefore conclude that the amino acid sequence of the beta 2-tubulin internal variable region is required for generation of correct axoneme morphology but not for general microtubule functions. As we have previously reported, the beta 2-tubulin carboxy terminal isotype-defining domain is required for suprastructural organization of the axoneme. We demonstrate here that the beta 2 variant lacking the carboxy terminus and the B2t6 variant complement each other for mild-to-moderate meiotic defects but do not complement for proper axonemal morphology. Our results are consistent with the hypothesis drawn from comparisons of vertebrate beta-tubulins that the two isotype-defining domains interact in a three-dimensional structure in wild-type beta-tubulins. We propose that the integrity of this structure in the Drosophila testis beta 2-tubulin isoform is required for proper axoneme assembly but not necessarily for general microtubule functions. On the basis of our observations we present a model for regulation of axoneme microtubule morphology as a function of tubulin assembly kinetics.

Ca2+-dependent interaction of S100A2 with muscle and nonmuscle tropomyosins

1997 ◽  
Vol 110 (5) ◽  
pp. 611-621 ◽  
Author(s):  
M. Gimona ◽  
Z. Lando ◽  
Y. Dolginov ◽  
J. Vandekerckhove ◽  
R. Kobayashi ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Epithelial Cell Line ◽  
Chemical Crosslinking ◽  
Related Function ◽  
Differentiated Cells ◽  
Cell Extracts ◽  
Sds Page ◽  
Functional Implications ◽  
Dependent Interaction

Zero-length chemical crosslinking with 1-ethyl-3-[3-(dimethyl amino)propyl]carbodiimide (EDC) indicated an association of the Ca2+-binding protein S100A2 with tropomyosin (TM) in vitro. The mobility of the crosslinked product on SDS-PAGE gels indicated the formation of a 1:1 complex between S100A2 and TM and the interaction was Ca2+ dependent. Monoclonal antibodies were raised against S100A2 and used to determine its cellular localization in the porcine epithelial cell line LLC PK1. It was found that the localization of S100A2 depended on the differentiation state of the cells, being absent from actin stress fibers in sparsely seeded cultures, but present in the actin-containing microvilli characteristic of differentiated cells. Immunoprecipitations of [35S]methionine-labeled extracts using S100A2 as well as TM-specific antibodies failed to co-precipitate TM and S100A2, indicating a transient association between these two molecules in solution. Affinity chromatography of cell extracts on immobilized recombinant TMs, however, confirmed the Ca2+-dependent interaction between S100A2 and both muscle TMs as well as with high and low molecular mass nonmuscle TMs, suggesting that the binding site resides in one of the conserved regions of TM. Our data demonstrate the possible interaction of S100A2 with TM that is not bound to the microfilaments and indicate a differentiation-related function for S100A2 in LLC PK1 cells. The possible functional implications of this interaction are discussed.

Sign in / Sign up

Export Citation Format

Share Document