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 Cytokinesis in Prokaryotes and Eukaryotes: Common Principles and Different Solutions

2001 ◽  
Vol 65 (2) ◽  
pp. 319-333 ◽  
Author(s):  
Nanne Nanninga
Keyword(s):  
Dna Replication ◽  
Cellular Structures ◽  
Microtubule Organizing Center ◽  
Microtubule Associated Proteins ◽  
Origin Of Dna Replication ◽  
Organizing Center ◽  
Associated Proteins ◽  
Eukaryotic Origin ◽  
Common Principle ◽  
Macromolecular Composition

SUMMARY Cytokinesis requires duplication of cellular structures followed by bipolarization of the predivisional cell. As a common principle, this applies to prokaryotes as well as eukaryotes. With respect to eukaryotes, the discussion has focused mainly on Saccharomyces cerevisiae and on Schizosaccharomyces pombe. Escherichia coli and to a lesser extent Bacillus subtilis have been used as prokaryotic examples. To establish a bipolar cell, duplication of a eukaryotic origin of DNA replication as well as its genome is not sufficient. Duplication of the microtubule-organizing center is required as a prelude to mitosis, and it is here that the dynamic cytoskeleton with all its associated proteins comes to the fore. In prokaryotes, a cytoskeleton that pervades the cytoplasm appears to be absent. DNA replication and the concomitant DNA segregation seem to occur without help from extensive cytosolic supramacromolecular assemblies but with help from the elongating cellular envelope. Prokaryotic cytokinesis proceeds through a contracting ring, which has a roughly 100-fold-smaller circumference than its eukaryotic counterpart. Although the ring contains proteins that can be considered as predecessors of actin, tubulin, and microtubule-associated proteins, its macromolecular composition is essentially different.

scholarly journals Daughter Centriole Elongation Is Controlled by Proteolysis

2010 ◽  
Vol 21 (22) ◽  
pp. 3942-3951 ◽  
Author(s):  
Nina Korzeniewski ◽  
Rolando Cuevas ◽  
Anette Duensing ◽  
Stefan Duensing
Keyword(s):  
Mammalian Cells ◽  
Gene Products ◽  
Microtubule Organizing Center ◽  
Future Studies ◽  
Microtubule Stability ◽  
Organizing Center ◽  
Pericentriolar Material ◽  
Substrate Cleavage ◽  
Interfering Rna ◽  
Daughter Centriole

The centrosome is the major microtubule-organizing center of most mammalian cells and consists of a pair of centrioles embedded in pericentriolar material. Before mitosis, the two centrioles duplicate and two new daughter centrioles form adjacent to each preexisting maternal centriole. After initiation of daughter centriole synthesis, the procentrioles elongate in a process that is poorly understood. Here, we show that inhibition of cellular proteolysis by Z-L3VS or MG132 induces abnormal elongation of daughter centrioles to approximately 4 times their normal length. This activity of Z-L3VS or MG132 was found to correlate with inhibition of intracellular protease-mediated substrate cleavage. Using a small interfering RNA screen, we identified a total of nine gene products that either attenuated (seven) or promoted (two) abnormal Z-L3VS–induced daughter centriole elongation. Our hits included known regulators of centriole length, including CPAP and CP110, but, interestingly, several proteins involved in microtubule stability and anchoring as well as centrosome cohesion. This suggests that nonproteasomal functions, specifically inhibition of cellular proteases, may play an important and underappreciated role in the regulation of centriole elongation. They also highlight the complexity of daughter centriole length control and provide a framework for future studies to dissect the molecular details of this process.

Identification of a 40 × 10(3) Mr centromere-associated protein in cultured mammalian cells

1990 ◽  
Vol 97 (4) ◽  
pp. 705-713
Author(s):  
R. Balczon ◽  
M.A. Accavitti ◽  
B.R. Brinkley
Keyword(s):  
Mammalian Cells ◽  
Cell Types ◽  
Immunoblot Analysis ◽  
Structure And Function ◽  
Interphase Nuclei ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Cytoplasmic Microtubules ◽  
And Function

Monoclonal antibodies were raised against a complex of proteins that was purified following the crosslinking of tubulin to the centromeres of CHO chromosomes using Lomant's reagent. One of the clones, hybridoma 32–9, produced antibodies that reacted with a 40 × 10(3) Mr protein present in the crosslinked complex. Furthermore, immunoblot analysis demonstrated that the 40 × 10(3) Mr antigen was present in various mammalian cell types from several different species. Indirect immunofluorescence using the antibody produced by clone 32–9 demonstrated that the 40 × 10(3) Mr antigen was associated with both spindle and cytoplasmic microtubules. In addition, centromere/kinetochore staining was detected in metaphase-arrested cells, while staining of prekinetochores in interphase nuclei was not observed. Unlike microtubule-associated proteins and microtubule-dependent ATPases, the 40 × 10(3) Mr protein did not copurify with microtubules when tubules were assembled from cellular homogenates using taxol and either GTP or GTP and AMP-PNP. Instead, the 40 × 10(3) Mr protein remained associated with the insoluble cellular material. The 40 × 10(3) Mr antigen could be released from the insoluble pelleted material by extraction with 1 M NaCl. Once solubilized, the 40 × 10(3) Mr protein was able to copurify with microtubules in assembly assays in vitro. This monoclonal antibody should serve as a valuable probe for studies of centromere/kinetochore structure and function.

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.

scholarly journals Mapmodulin, Cytoplasmic Dynein, and Microtubules Enhance the Transport of Mannose 6-Phosphate Receptors from Endosomes to theTrans-Golgi Network

1999 ◽  
Vol 10 (7) ◽  
pp. 2191-2197 ◽  
Author(s):  
Christian Itin ◽  
Nirit Ulitzur ◽  
Bettina Mühlbauer ◽  
Suzanne R. Pfeffer
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Cytoplasmic Dynein ◽  
Vesicle Transport ◽  
Microtubule Associated Proteins ◽  
Late Endosomes ◽  
Associated Proteins ◽  
Mannose 6 Phosphate ◽  
Golgi Network

Late endosomes and the Golgi complex maintain their cellular localizations by virtue of interactions with the microtubule-based cytoskeleton. We study the transport of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network in vitro. We show here that this process is facilitated by microtubules and the microtubule-based motor cytoplasmic dynein; transport is inhibited by excess recombinant dynamitin or purified microtubule-associated proteins. Mapmodulin, a protein that interacts with the microtubule-associated proteins MAP2, MAP4, and tau, stimulates the microtubule- and dynein-dependent localization of Golgi complexes in semi-intact Chinese hamster ovary cells. The present study shows that mapmodulin also stimulates the initial rate with which mannose 6-phosphate receptors are transported from late endosomes to thetrans-Golgi network in vitro. These findings represent the first indication that mapmodulin can stimulate a vesicle transport process, and they support a model in which the microtubule-based cytoskeleton enhances the efficiency of vesicle transport between membrane-bound compartments in mammalian cells.

subito Encodes a Kinesin-like Protein Required for Meiotic Spindle Pole Formation in Drosophila melanogaster

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1489-1501 ◽  
Author(s):  
Kelly L Giunta ◽  
Janet K Jang ◽  
Elizabeth A Manheim ◽  
Gayathri Subramanian ◽  
Kim S McKim
Keyword(s):  
Drosophila Melanogaster ◽  
Spindle Pole ◽  
Meiotic Spindle ◽  
Microtubule Organizing Center ◽  
Bipolar Spindle ◽  
Microtubule Associated Proteins ◽  
Organizing Center ◽  
Associated Proteins ◽  
Similar Phenotype ◽  
Meiosis I

Abstract The female meiotic spindle lacks a centrosome or microtubule-organizing center in many organisms. During cell division, these spindles are organized by the chromosomes and microtubule-associated proteins. Previous studies in Drosophila melanogaster implicated at least one kinesin motor protein, NCD, in tapering the microtubules into a bipolar spindle. We have identified a second Drosophila kinesin-like protein, SUB, that is required for meiotic spindle function. At meiosis I in males and females, sub mutations affect only the segregation of homologous chromosomes. In female meiosis, sub mutations have a similar phenotype to ncd; even though chromosomes are joined by chiasmata they fail to segregate at meiosis I. Cytological analyses have revealed that sub is required for bipolar spindle formation. In sub mutations, we observed spindles that were unipolar, multipolar, or frayed with no defined poles. On the basis of these phenotypes and the observation that sub mutations genetically interact with ncd, we propose that SUB is one member of a group of microtubule-associated proteins required for bipolar spindle assembly in the absence of the centrosomes. sub is also required for the early embryonic divisions but is otherwise dispensable for most mitotic divisions.

scholarly journals Drosophila gamma-tubulin is part of a complex containing two previously identified centrosomal MAPs.

1993 ◽  
Vol 121 (4) ◽  
pp. 823-835 ◽  
Author(s):  
J W Raff ◽  
D R Kellogg ◽  
B M Alberts
Keyword(s):  
Microtubule Organizing Center ◽  
Substantial Fraction ◽  
Beta Tubulin ◽  
Microtubule Associated Proteins ◽  
Organizing Center ◽  
Associated Proteins ◽  
Gamma Tubulin ◽  
A Minor ◽  
Drosophila Embryos

gamma-tubulin is a minor tubulin that is localized to the microtubule organizing center of many fungi and higher eucaryotic cells (Oakley, B. R., C. E. Oakley, Y. Yoon, and M. C. Jung. 1990. Cell. 61: 1289-1301; Stearns, T., L. Evans, and M. Kirschner. 1991. Cell. 65:825-836; Zheng, Y., M. K. Jung, and B. R. Oakley. 1991. Cell. 65:817-823). Here we show that gamma-tubulin is a component of a previously isolated complex of Drosophila proteins that contains at least two centrosomal microtubule-associated proteins called DMAP190 and DMAP60. Like DMAP190 and DMAP60, the gamma-tubulin in extracts of early Drosophila embryos binds to microtubules, although this binding may be indirect. Unlike DMAP190 and DMAP60, however, only 10-50% of the gamma-tubulin in the extract is able to bind to microtubules. We show that gamma-tubulin binds to a microtubule column as part of a complex, and a substantial fraction of this gamma-tubulin is tightly associated with DMAP60. As neither alpha- nor beta-tubulin bind to microtubule columns, the gamma-tubulin cannot be binding to such columns in the form of an alpha:gamma or beta:gamma heterodimer. These observations suggest that gamma-tubulin, DMAP60, and DMAP190 are components of a centrosomal complex that can interact with microtubules.

scholarly journals Dynamic interactions of fluorescently labeled microtubule-associated proteins in living cells.

1984 ◽  
Vol 99 (2) ◽  
pp. 425-434 ◽  
Author(s):  
T Scherson ◽  
T E Kreis ◽  
J Schlessinger ◽  
U Z Littauer ◽  
G G Borisy ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Dynamic Properties ◽  
Cell Types ◽  
Recovery Phase ◽  
Slow Recovery ◽  
Double Labeling ◽  
Microtubule Associated Proteins ◽  
Associated Proteins

Microtubule-associated proteins (MAPs) from calf brain were fluorescently labeled with 6-iodoacetamido fluorescein (I-AF). The modified MAPs (especially enriched for MAP2) were fully active in promoting tubulin polymerization in vitro and readily associated with cytoplasmic filaments when microinjected into living cultured cells. Double-labeling experiments indicated that the microinjected AF-MAPs were incorporated predominantly, if not exclusively, into cytoplasmic microtubules in untreated cells or paracrystals induced within vinblastine-treated cells. Similar results were obtained with different cell types (neuronal, epithelial, and fibroblastic) of diverse origin (man, mouse, chicken, and rat kangaroo). Mobility measurements of the microinjected AF-MAPs using the method of fluorescence-photobleaching recovery (FPR) revealed two populations of AF-MAPs with distinct dynamic properties: One fraction represents the soluble pool of MAPs and is mobile with a diffusion coefficient of D = 3 X 10(-9) cm2/s. The other fraction of MAPs is associated with the microtubules and is essentially immobile on the time scale of FPR experiments. However, it showed slow fluorescence recovery with an apparent half time of approximately 5 min. The slow recovery of fluorescence on defined photobleached microtubules occurred most probably by the incorporation of AF-MAPs from the soluble cytoplasmic pool into the bleached area. The bleached spot on defined microtubules remained essentially immobile during the slow recovery phase. These results suggest that MAPs can associate in vivo with microtubules of diverse cell types and that treadmilling of MAP2-containing microtubules in vivo, if it exists, is slower than 4 micron/h.

scholarly journals PLK4 is a microtubule-associated protein that self assembles promoting de novo MTOC formation

2018 ◽  
Author(s):  
Susana Montenegro Gouveia ◽  
Sihem Zitouni ◽  
Dong Kong ◽  
Paulo Duarte ◽  
Beatriz Ferreira Gomes ◽  
...  
Keyword(s):  
De Novo ◽  
Supramolecular Assemblies ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
Organizing Center ◽  
Pericentriolar Material ◽  
Xenopus Egg ◽  
Summary Statement ◽  
Β Tubulin

Summary statementPLK4 binds to microtubules and self assembles into supramolecular assemblies that recruit tubulin and trigger de novo MTOC formation in Xenopus laevis extracts.AbstractThe centrosome is an important microtubule-organizing center (MTOCs) in animal cells and it consists of two barrel-shaped structures (centrioles), surrounded by the pericentriolar material (PCM), which nucleates microtubules. PCM components form condensates, supramolecular assemblies that concentrate microtubule nucleators. Centrosomes can form close to an existing structure (canonical duplication) or de novo. How centrosomes form de novo is not known. PLK4 is a master driver of centrosome biogenesis, which is critical to recruit several centriole components. Here, we investigate the beginning of centrosome biogenesis, taking advantage of Xenopus egg extracts, where we and others have shown that PLK4 can induce de novo MTOC formation (Eckerdt et al., 2011; Zitouni et al., 2016). Surprisingly, we observe that in vitro, PLK4 can self-assemble into supramolecular assemblies that recruit α/β-tubulin. In Xenopus extracts, PLK4 supramolecular assemblies additionally recruit the PLK4 substrate STIL and the microtubule nucleator, γ-tubulin, and form acentriolar MTOCs de novo. The assembly of these robust microtubule asters is independent of dynein, similarly to centrosomes. We suggest a new mechanism of action for PLK4, where it forms a self-organizing catalytic scaffold that recruits centriole components, PCM factors and α/β-tubulin, leading to MTOC formation.

Evolution of tools and methods for monitoring autophagic flux in mammalian cells

2018 ◽  
Vol 46 (1) ◽  
pp. 97-110 ◽  
Author(s):  
Kevin C. Yang ◽  
Paalini Sathiyaseelan ◽  
Cally Ho ◽  
Sharon M. Gorski
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Autophagic Flux ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
A Cell ◽  
Degradation Step ◽  
Autophagy Pathway ◽  
Practice Methods ◽  
Tools And Methods

Autophagy is an evolutionarily conserved lysosome-mediated degradation and recycling process, which functions in cellular homeostasis and stress adaptation. The process is highly dynamic and involves autophagosome synthesis, cargo recognition and transport, autophagosome–lysosome fusion, and cargo degradation. The multistep nature of autophagy makes it challenging to quantify, and it is important to consider not only the number of autophagosomes within a cell but also the autophagic degradative activity. The rate at which cargos are recognized, segregated, and degraded through the autophagy pathway is defined as autophagic flux. In practice, methods to measure autophagic flux typically evaluate the lysosome-mediated cargo degradation step by leveraging known autophagy markers such as MAP1LC3B (microtubule-associated proteins 1A/1B light chain 3 beta) or lysosome-dependent fluorescent agents. In this review, we summarize the tools and methods used in mammalian cultured cells pertaining to these two approaches, and highlight innovations that have led to their evolution in recent years. We also discuss the potential limitations of these approaches and recommend using a combination of strategies and multiple different autophagy markers to reliably evaluate autophagic flux in mammalian cells.

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