scholarly journals Calmodulin-microtubule association in cultured mammalian cells.

1984 ◽  
Vol 98 (3) ◽  
pp. 904-910 ◽  
Author(s):  
W J Deery ◽  
A R Means ◽  
B R Brinkley
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Cell System ◽  
The Other ◽  
Microtubule Assembly ◽  
Cytoplasmic Microtubule ◽  
Hypotonic Treatment ◽  
Cytoplasmic Microtubules ◽  
Triton X ◽  
Ca Sensitivity

A Triton X-100-lysed cell system has been used to identify calmodulin on the cytoskeleton of 3T3 and transformed SV3T3 cells. By indirect immunofluorescence, calmodulin was found to be associated with both the cytoplasmic microtubule complex and the centrosomes. A number of cytoplasmic microtubules more resistant to disassembly upon either cold (0-4 degrees C) or hypotonic treatment, as well as following dilution have been identified. Most of the stable microtubules appeared to be associated with the centrosome at one end and with the plasma membrane at the other end. These microtubules could be induced to depolymerize, however, by micromolar Ca++ concentrations. These data suggest that, by interacting directly with the microtubule, calmodulin may influence microtubule assembly and ensure the Ca++-sensitivity of both mitotic and cytoplasmic microtubules.

scholarly journals Organization of the flagellar apparatus and associate cytoplasmic microtubules in the quadriflagellate alga Polytomella agilis.

1976 ◽  
Vol 69 (1) ◽  
pp. 106-125 ◽  
Author(s):  
D L Brown ◽  
A Massalski ◽  
R Patenaude
Keyword(s):  
Anterior Part ◽  
Flagellar Apparatus ◽  
Microtubule Assembly ◽  
Immunofluorescent Staining ◽  
Body Region ◽  
Basal Bodies ◽  
Cytoplasmic Microtubule ◽  
Large Numbers ◽  
Attachment Sites ◽  
Cytoplasmic Microtubules

The organization of microtubular systems in the quadriflagellate unicell Polytomella agilis has been reconstructed by electron microscopy of serial sections, and the overall arrangement confirmed by immunofluorescent staining using antiserum directed against chick brain tubulin. The basal bodies of the four flagella are shown to be linked in two pairs of short fibers. Light microscopy of swimming cells indicates that the flagella beat in two synchronous pairs, with each pair exhibiting a breast-stroke-like motion. Two structurally distinct flagellar rootlets, one consisting of four microtubules in a 3 over 1 pattern and the other of a striated fiber over two microtubules, terminate between adjacent basal bodies. These rootlets diverge from the basal body region and extend toward the cell posterior, passing just beneath the plasma membrane. Near the anterior part of the cell, all eight rootlets serve as attachment sites for large numbers of cytoplasmic microtubules which occur in a single row around the circumference of the cell and closely parallel the cell shape. It is suggested that the flagellar rootless may function in controlling the patterning and the direction of cytoplasmic microtubule assembly. The occurrence of similar rootlet structures in other flagellates is briefly reviewed.

scholarly journals Human chromosomes and centrioles as nucleating sites for the in vitro assembly of microtubules from bovine brain tubulin.

1975 ◽  
Vol 67 (1) ◽  
pp. 189-199 ◽  
Author(s):  
M McGill ◽  
B R Brinkley
Keyword(s):  
Mammalian Cells ◽  
Experimental Studies ◽  
Bovine Brain ◽  
Microtubule Assembly ◽  
Direct Result ◽  
Metaphase Chromosomes ◽  
Microtubule Formation ◽  
Triton X ◽  
Brain Tubulin

Treatment of HeLa cells with Colcemid at concentrations of 0.06-0.10 mug/ml leads to irreversible arrest in mitosis. Colcemid-arrested cells contained few microtubules, and many kinetochores and centrioles were free of microtubule association. When these cells were exposed to microtubule reassembly buffer containing Triton X-100 and bovine brain tubulin at 37 degrees C, numerous microtubules were reassembled at all kinetochores of metaphase chromosomes and in association with centriole pairs. When bovine brain tubulin was eliminated from the reassembly system, microtubules failed to assemble at these sites. Similarly, when EGTA was eliminated from the reassembly system, microtubules failed to polymerize. These results are consistent with other investigations of in vitro microtubule assembly and indicate that HeLa chromosomes and centrioles can serve as nucleating sites for the assembly of microtubules from brain tubulin. Both chromosomes and centrioles became displaced from their C-metaphase configurations during tubulin reassembly, indicating that their movements were a direct result of microtubule formation. Although both kinetochore- and centriole-associated microtubules were assembled and movement occurred, we did not observe direct extension of microtubules from kinetochores to centrioles. This system should prove useful for experimental studies of spindle microtubule formation and chromosome movement in mammalian cells.

scholarly journals Purification of Lipid Rafts from Cultured Cells

2002 ◽  
Vol 2 ◽  
pp. 1662-1666 ◽  
Author(s):  
John Graham
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Microsomal Fraction ◽  
Cultured Cells ◽  
The Other ◽  
Low Concentrations ◽  
Nonionic Detergent ◽  
Triton X ◽  
Intact Lipid

Lipid-rich lipid rafts are microdomains of the plasma membrane that are resistant to low concentrations of nonionic detergent. This forms the basis for their isolation. Either a microsomal fraction or a postnuclear supernatant are loaded beneath a discontinuous iodixanol gradient. If all the solutions contain 0.5–1.0% Triton X-100, the intact lipid rafts float to the top of the gradient while all of the other detergent-solubilized membranes remain at the bottom.

scholarly journals A new class of Paramecium surface proteins anchored in the plasma membrane by a glycosylinositol phospholipid. Membrane anchor of Paramecium cross-reacting glycoproteins

1988 ◽  
Vol 253 (2) ◽  
pp. 395-400 ◽  
Author(s):  
C Deregnaucourt ◽  
A M Keller ◽  
Y Capdeville
Keyword(s):  
Plasma Membrane ◽  
Surface Proteins ◽  
The Other ◽  
Phospholipid Membrane ◽  
Membrane Anchor ◽  
New Class ◽  
Ethanolic Extracts ◽  
Covalently Linked ◽  
Triton X ◽  
Variant Surface Glycoproteins

Treatment of paramecia with ethanol or Triton X-100 solubilizes a major membrane protein, namely the surface antigen (SAg), and a set of glycopeptides in the range 40-60 kDa, which cross-react with the SAg. We demonstrate that these glycopeptides, called ‘cross-reacting glycoproteins’ (CRGs), are distinct molecules from the SAg. First, after purification of CRGs from ethanolic extracts of Paramecium primaurelia expressing the 156G SAg, the amino acid composition of a given CRG was found to be different from, and incompatible with, that of the 156G SAg. Secondly, we showed that the CRGs, although not immunologically detectable, are present in fractions containing the myristoylated form of the 156G SAg. The treatment of these fractions by phosphatidylinositol-specific phospholipases C enables us to reveal the CRGs through the unmasking of two distinct epitopes. One is the ‘cross-reacting determinant’ (CRD), initially described for the variant surface glycoproteins (VSGs) of Trypanosoma; the other determinant, called ‘det-2355’, is specific to the SAg and to the CRGs. Our results suggest that (1) phosphatidylinositol is covalently linked to the CRGs and (2) the CRD and the det-2355 are localized in the same region of the CRGs. We propose that the CRGs are a new set of surface proteins anchored in the cell membrane of Paramecium via a glycosylinositol phospholipid, in the same way as the SAgs.

scholarly journals Distance-dependent cellular palmitoylation of de-novo-designed sequences and their translocation to plasma membrane subdomains

2002 ◽  
Vol 115 (15) ◽  
pp. 3119-3130 ◽  
Author(s):  
Inmaculada Navarro-Lérida ◽  
Alberto Álvarez-Barrientos ◽  
Francisco Gavilanes ◽  
Ignacio Rodriguez-Crespo
Keyword(s):  
Plasma Membrane ◽  
Protein Sequence ◽  
Mammalian Cells ◽  
De Novo ◽  
Cysteine Residue ◽  
Dependent Manner ◽  
Caveolin 1 ◽  
Protein Palmitoylation ◽  
Triton X

Using recursive PCR, we created an artificial protein sequence that consists of a consensus myristoylation motif (MGCTLS) followed by the triplet AGS repeated nine times and fused to the GFP reporter. This linker-GFP sequence was utilized as a base to produce multiple mutants that were used to transfect COS-7 cells. Constructs where a `palmitoylable' cysteine residue was progressively moved apart from the myristoylation site to positions 3, 9, 15 and 21 of the protein sequence were made, and these mutants were used to investigate the effect of protein myristoylation on subsequent palmitoylation,subcellular localization, membrane association and caveolin-1 colocalization. In all cases, dual acylation of the GFP chimeras correlated with translocation to Triton X-100-insoluble cholesterol/sphingomyelin-enriched subdomains. Whereas a strong Golgi labeling was observed in all the myristoylated chimeras, association with the plasma membrane was only observed in the dually acylated constructs. Taking into account the conflicting data regarding the existence and specificity of cellular palmitoyl-transferases, our results provide evidence that de-novo-designed sequences can be efficiently S-acylated with palmitic acid in vivo, strongly supporting the hypothesis that non-enzymatic protein palmitoylation can occur within mammalian cells. Additionally, this palmitoylation results in the translocation of the recombinant construct to low-fluidity domains in a myristate-palmitate distance-dependent manner.

Plasma membrane clustering of system y+ (CAT-1) amino acid transporter as detected by immunohistochemistry

1994 ◽  
Vol 266 (5) ◽  
pp. E817-E824 ◽  
Author(s):  
M. H. Woodard ◽  
W. A. Dunn ◽  
R. O. Laine ◽  
M. Malandro ◽  
R. McMahon ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Human Fibroblasts ◽  
Amino Acid Transporter ◽  
Microtubule Assembly ◽  
Random Pattern ◽  
Transporter Protein ◽  
Acid Transporter

Transport of cationic amino acids in fully differentiated mammalian cells is mediated primarily by system y1+ [cationic amino acid transporter (CAT)-1 gene product]. Antibodies, prepared against synthetic peptide sequences predicted to be extracellular loops of the CAT-1 transporter protein, detected the transporter on the surface of cultured cells. In human fibroblasts, porcine pulmonary artery endothelial cells, and cultured rat hepatoma cells, the CAT-1 transporter protein was clustered in an apparent random pattern throughout the plasma membrane. In contrast, labeling of the fibroblasts with antibodies against the epidermal growth factor receptor or the GLUT-1 glucose transporter demonstrated a uniform staining pattern covering the entire cell surface. The CAT-1 antibody labeling was specific, as demonstrated by peptide inhibition and the lack of staining by preimmune serum. Furthermore, hepatocytes did not exhibit specific antibody binding consistent with the lack of system y1+ activity. Disruption of the microtubule assembly resulted in a reversible loss of the CAT-1 transporter clusters and a more generalized labeling of the cell body. The data demonstrate the existence of microdomains within the plasma membrane that contain the CAT-1 transporter protein.

scholarly journals Tubulin assembly sites and the organization of cytoplasmic microtubules in cultured mammalian cells.

1981 ◽  
Vol 90 (3) ◽  
pp. 554-562 ◽  
Author(s):  
B R Brinkley ◽  
S M Cox ◽  
D A Pepper ◽  
L Wible ◽  
S L Brenner ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Simian Virus 40 ◽  
Spatial Arrangement ◽  
Simian Virus ◽  
Microtubule Assembly ◽  
Daughter Cells ◽  
Pericentriolar Material ◽  
Cytoplasmic Microtubules ◽  
Kinetics Of

The number, distribution, and nucleating capacity of microtubule-organizing centers (MTOCs) has been investigated in a variety of cultured mammalian cells. Most interphase cells contain a single MTOC that is localized at the centrosome region and corresponds to the centriole and pericentriolar material. MTOCs, like centrioles, become duplicated during the S phase of the cell cycle and are equationally distributed to daughter cells in mitosis. Multiple MTOCs were rarely observed in cultured cells except in one cell line (neuroblastoma), which also displayed an equally large number of centrioles in the cytoplasm. The kinetics of microtubule assembly and the tubulin nucleating capacity of MTOCs was assayed by incubating tubulin-depleted, permeabilized 3T3 and simian virus 40-transformed 3T3 cells with phosphocellulose-purified 65 brain tubulin and microtubule assembly buffer. Initiation and assembly of 65 tubulin occurred in association with the cells' endogenous MTOCs, and the length, number, and distribution of microtubules generated about the organizing centers were regulated and cell specific. Our results are consistent with the notion that the specification of microtubule length, number, and spatial arrangement resides largely in the MTOCs and surrounding cytoplasm and not in the tubulin subunits.

Regulation of Microtubule Assembly-Disassembly in Mitotic Mammalian Cells: Role of Calcium, Calmodulin and MTOCs

1981 ◽  
Vol 39 ◽  
pp. 572-575
Author(s):  
B. R. Brinkley ◽  
S. L. Brenner ◽  
D. A. Pepper ◽  
R. L. Pardue
Keyword(s):  
Cell Cycle ◽  
Calcium Binding ◽  
Mammalian Cells ◽  
Microtubule Assembly ◽  
Free Calcium ◽  
Mitotic Apparatus ◽  
Cytoplasmic Microtubule ◽  
Dividing Cells ◽  
Late Telophase

Two microtubule arrays exist in cultured mammalian cells during their progression through the cell cycle; the cytoplasmic microtubule complexes (CMTC) of interphase cells (Figure 1) and the mitotic apparatus (MA) of dividing cells (Figure 2). As chromosomes are segregated to opposite poles of the spindle during telophase, the microtubules of the MA are disassembled. During late telophase -G1 phase the tubulin subunits from the spindle are recycled into the microtubules of the CMTC which forms an elaborate network throught the cytoplasm. When cells progress into late G2 -Prophase, the CMTC is disassembled and the tubulin is converted into microtubules of the MA. our research has been aimed at defining the mechanism whereby cells regulate the alternating patterns of microtubule assembly-disassembly during the cell cycle.In one series of experiments, we have investigated the role of calcium in microtubule assembly. Several laboratories have shown that cytoplasmic and spindle microtubules are unstable in the presence of elevated free calcium levels. Using monospecific antibodies and indirect immunofluorescence, we have demonstrated the presence of the ubiquitous calcium-binding protein calmodulin in the mitotic spindle of mammalian cells in vitro (Figure 3).

scholarly journals Cytoplasmic microtubule assembly-disassembly from endogenous tubulin in a Brij-lysed cell model.

1983 ◽  
Vol 96 (6) ◽  
pp. 1631-1641 ◽  
Author(s):  
W J Deery ◽  
B R Brinkley
Keyword(s):  
Cell Model ◽  
Living Cells ◽  
Cell System ◽  
Significant Fraction ◽  
Microtubule Assembly ◽  
Cytoplasmic Microtubule ◽  
Brij 58 ◽  
Cold Sensitive

We studied the characteristics of cytoplasmic microtubule reassembly from endogenous tubulin pools in situ using a Brij 58-lysed 3T3 cell system. Cells that were pretreated in vivo with colcemid retain endogenous tubulin in the depolymerized state after lysis. When lysed cells were removed from colcemid block and incubated in GTP-PIPES reassembly buffer at pH 6.9, microtubules repolymerized randomly throughout the cytoplasm, appeared to be free-ended and were generally not associated with the centrosomes. However, tubulin could be induced to polymerize in an organized manner from the centrosomes by increasing the pH to 7.6 in the presence of ATP and cAMP. Microtubules polymerized in ATP had significantly longer lengths than those assembled in GTP or UTP. When cells not treated with colcemid were lysed, the integrity of the cytoplasmic microtubule complex (CMTC) was maintained during subsequent incubation in reassembly buffer. However, in contrast to unlysed, living cells, microtubules of lysed cells were stable to colchicine. A significant fraction of the CMTC was stable to cold-induced disassembly whereas microtubules reassembled after lysis were extremely cold-sensitive. When cells not treated with colcemid were lysed and incubated in millimolar Ca++, microtubules depolymerized from their distal ends and a much reduced CMTC was observed. Ca++ reversal with EGTA rapidly resulted in a reformation of the CMTC apparently by elongation of Ca++ resistant microtubules.

scholarly journals Microtubule initiation at kinetochores and centrosomes in lysed mitotic cells. Inhibition of site-specific nucleation by tubulin antibody.

1979 ◽  
Vol 82 (2) ◽  
pp. 585-591 ◽  
Author(s):  
D A Pepper ◽  
B R Brinkley
Keyword(s):  
Electron Microscopy ◽  
Cell System ◽  
Molecular Composition ◽  
Microtubule Assembly ◽  
Microtubule Nucleation ◽  
Site Specific ◽  
Subsequent Exposure ◽  
Triton X ◽  
Mitotic Cells

A lysed cell system was developed to determine whether tubulin antibody can block the nucleation of exogenous tubulin at kinetochores and centrosomes. Mitotic PtK2 cells were pretreated with colcemid to remove all endogenous microtubules and were lysed with Triton X-100 in PIPES-EGTA-Mg++ buffer. This procedure left centrosomes, chromosomes, and kinetochores intact as determined by electron microscopy of thin-sectioned cells. Exposure of the lysed cells to phorphocellulose-purified tubulin dimers at 37 degrees C in the presence of 1 mM GTP resulted in site-specific nucleation of microtubules at centrosomes and kinetochores. Treatment of the lysed cell preparations with tubulin antibody before subsequent exposure to the exogenous tubulin resulted in almost complete blockage of microtubule nucleation, especially at kinetochores. Pretreatment of the lysed cell preparations with control antibody or buffer without antibody had no effect on the ability of centrosomes and kinetochores to initiate microtubule assembly. The implications of these results with respect to the molecular composition of centrosomes and kinetochores are discussed.

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