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.

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.

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 TAPping into the treasures of tubulin using novel protein production methods

2018 ◽  
Vol 62 (6) ◽  
pp. 781-792
Author(s):  
Nuo Yu ◽  
Niels Galjart
Keyword(s):  
Mammalian Cells ◽  
Gene Families ◽  
Cell Types ◽  
Cellular Functions ◽  
Tubulin Isotypes ◽  
Associated Proteins ◽  
Cytoskeletal Elements ◽  
Different Cell Types ◽  
Β Tubulin

Microtubules are cytoskeletal elements with important cellular functions, whose dynamic behaviour and properties are in part regulated by microtubule-associated proteins (MAPs). The building block of microtubules is tubulin, a heterodimer of α- and β-tubulin subunits. Longitudinal interactions between tubulin dimers facilitate a head-to-tail arrangement of dimers into protofilaments, while lateral interactions allow the formation of a hollow microtubule tube that mostly contains 13 protofilaments. Highly homologous α- and β-tubulin isotypes exist, which are encoded by multi-gene families. In vitro studies on microtubules and MAPs have largely relied on brain-derived tubulin preparations. However, these consist of an unknown mix of tubulin isotypes with undefined post-translational modifications. This has blocked studies on the functions of tubulin isotypes and the effects of tubulin mutations found in human neurological disorders. Fortunately, various methodologies to produce recombinant mammalian tubulins have become available in the last years, allowing researchers to overcome this barrier. In addition, affinity-based purification of tagged tubulins and identification of tubulin-associated proteins (TAPs) by mass spectrometry has revealed the ‘tubulome’ of mammalian cells. Future experiments with recombinant tubulins should allow a detailed description of how tubulin isotype influences basic microtubule behaviour, and how MAPs and TAPs impinge on tubulin isotypes and microtubule-based processes in different cell types.

scholarly journals Plk Phosphorylation Regulates the Microtubule-Stabilizing Protein TCTP

2002 ◽  
Vol 22 (17) ◽  
pp. 6209-6221 ◽  
Author(s):  
Frederic R. Yarm
Keyword(s):  
Protein A ◽  
Phosphorylation Site ◽  
Mitotic Cell ◽  
Structural Protein ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Mutant Phenotypes ◽  
And Function

ABSTRACT The mitotic polo-like kinases have been implicated in the formation and function of bipolar spindles on the basis of their respective localizations and mutant phenotypes. To date, this putative regulation has been limited to a kinesin-like motor protein, a centrosomal structural protein, and two microtubule-associated proteins (MAPs). In this study, another spindle-regulating protein, the mammalian non-MAP microtubule-binding and -stabilizing protein, the translationally controlled tumor protein (TCTP), was identified as a putative Plk-interacting clone by a two-hybrid screen. Plk phosphorylates TCTP on two serine residues in vitro and cofractionates with the majority of kinase activity toward TCTP in mitotic cell lysates. In addition, these sites were demonstrated to be phosphorylated in vivo. Overexpression of a Plk phosphorylation site-deficient mutant of TCTP induced a dramatic increase in the number of multinucleate cells, rounded cells with condensed ball-like nuclei, and cells undergoing cell death, similar to both the reported anti-Plk antibody microinjection and the low-concentration taxol treatment phenotypes. These results suggest that phosphorylation decreases the microtubule-stabilizing activity of TCTP and promotes the increase in microtubule dynamics that occurs after metaphase.

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.

Modeling of Artificial 3D Human Placenta

2021 ◽  
pp. 1-10
Author(s):  
Rumeysa Tutar ◽  
Betül Çelebi-Saltik
Keyword(s):  
Human Placenta ◽  
Placental Transfer ◽  
Complex Structure ◽  
Cell Types ◽  
Structure And Function ◽  
Technological Advances ◽  
And Function ◽  
Different Cell Types ◽  
Pregnancy And Birth

The placenta is the main organ that allows the fertilized oocyte to develop and mature. It allows the fetus to grow in the prenatal period by transferring oxygen and nutrients between the mother and the fetus. It acts as a basic endocrine organ which creates the physiological changes related to pregnancy and birth in the mother. Removal of wastes and carbon dioxide from the fetus is also achieved by the placenta. It prevents the rejection of the fetus and protects the fetus from harmful effects. Research on the human placenta focuses on understanding the placental structure and function to illuminate the complex structure of this important organ with technological advances. The structure and function of the placental barrier have been investigated with in vitro studies in 2D/3D, and various results have been published comparatively. In this review, we introduce the nature of the placenta with its 3D composition which has been called niche. Different cell types and placental structures are presented. We describe the systems and approaches used in the creation of current 3D placenta, placental transfer models as 3D placental barriers, and micro-engineered 3D placenta on-a-chip to explore complicated placental responses to nanoparticle exposure.

Microvascular Structure and Function in Vitro

2009 ◽  
Author(s):  
Abraham D. Stroock ◽  
Nak Won Choi ◽  
Tobias D. Wheeler ◽  
Valerie Cross ◽  
Scott Verbridge ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Structure And Function ◽  
Three Dimensions ◽  
Vascular Structure ◽  
Biological Applications ◽  
Convective Mass Transfer ◽  
Negative Pressures ◽  
And Function ◽  
And Control

Vascular structure — a network of convective paths — is a ubiquitous element in multicellular, living systems. The key function of vascular structure in animals and plants is mediation of convective mass transfer over macroscopic distances; this transfer allows an organism to monitor and control the chemical state of its tissues. In our laboratory, we are developing methods to embed and operate microfluidic systems within tissue-like materials in order to capture this function for both biological and non-biological applications. I will present two examples to illustrate our efforts: 1) Capillary beds for the culture of mammalian cells in three-dimensions. In this section, I will discuss the development of methods both to fabricate synthetic capillary beds and to grow them directly out of endothelial cells. I will highlight how simple ideas from continuum mechanics and material science have guided our efforts. 2) Synthetic xylem networks that allow for the transpiration of water at large negative pressures. I will point out the unusual thermodynamic and transport phenomena that are involved in the transpiration process in plants. I will then present our perspectives on the design criteria for systems — synthetic and biological — that mediate this process. Finally, I will describe our experiments with “synthetic trees” in which we have reproduced the main features of transpiration. I will conclude with perspectives on applications and generalizations of both these classes of vascularized materials.

scholarly journals Influence of phosphorylation on isoform composition and function of a microtubule-associated protein from developing Artemia

1995 ◽  
Vol 307 (2) ◽  
pp. 419-424 ◽  
Author(s):  
J Zhang ◽  
T H Macrae
Keyword(s):  
Brine Shrimp ◽  
Electrophoretic Analysis ◽  
Immunofluorescent Staining ◽  
Mobility Shift ◽  
Western Blots ◽  
Microtubule Associated Proteins ◽  
Apparent Homogeneity ◽  
Associated Proteins ◽  
And Function

A novel 49 kDa protein, which exhibits nucleotide-dependent cross-linking of microtubules in vitro and localizes to ordered microtubule arrays by immunofluorescent staining, has been purified to apparent homogeneity from the brine shrimp, Artemia. Electrophoretic analysis involving isoelectric focusing and two-dimensional gels, supplemented by staining of Western blots with affinity-purified antibody, revealed that the 49 kDa protein consists of five isoforms with pI values of 6.0-6.2. The amount of 49 kDa protein increased slightly, but its isoform composition did not change significantly, during development of Artemia gastrula to third-instar larvae. Treatment with alkaline phosphatase caused the 49 kDa protein to undergo a mobility shift on gel electrophoresis, and, by use of an antibody to phosphoserine, at least two isoforms of the protein were shown to be phosphorylated. The serine phosphate, presumably added by a post-translational mechanism, did not influence binding of the 49 kDa protein to microtubules. Under conditions in which microtubules were cross-linked, the 49 kDa protein failed to interact with actin filaments. Our results demonstrate that the 49 kDa protein, like other structural microtubule-associated proteins such as tau and MAP2, is composed of several isoforms, some of which are phosphorylated. This protein has the potential to regulate the spatial distribution of microtubules within cells but does not link microfilaments to one another or to microtubules.

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