scholarly journals Modulation of phosphatidylinositol 4-phosphate levels by CaBP7 controls cytokinesis in mammalian cells

2015 ◽  
Vol 26 (8) ◽  
pp. 1428-1439 ◽  
Author(s):  
Dayani Rajamanoharan ◽  
Hannah V. McCue ◽  
Robert D. Burgoyne ◽  
Lee P. Haynes
Keyword(s):  
Cell Division ◽  
Hela Cells ◽  
Calcium Binding ◽  
Mammalian Cells ◽  
Calcium Binding Protein ◽  
Model Systems ◽  
Intercellular Bridge ◽  
Phosphoinositide Signaling ◽  
Novel Association ◽  
Phosphatidylinositol 4

Calcium and phosphoinositide signaling regulate cell division in model systems, but their significance in mammalian cells is unclear. Calcium-binding protein-7 (CaBP7) is a phosphatidylinositol 4-kinaseIIIβ (PI4KIIIβ) inhibitor required during cytokinesis in mammalian cells, hinting at a link between these pathways. Here we characterize a novel association of CaBP7 with lysosomes that cluster at the intercellular bridge during cytokinesis in HeLa cells. We show that CaBP7 regulates lysosome clustering and that PI4KIIIβ is essential for normal cytokinesis. CaBP7 depletion induces lysosome mislocalization, extension of intercellular bridge lifetime, and cytokinesis failure. These data connect phosphoinositide and calcium pathways to lysosome localization and normal cytokinesis in mammalian cells.

scholarly journals A Myristoylated Calcium-binding Protein that Preferentially Interacts with the Alzheimer's Disease Presenilin 2 Protein

1999 ◽  
Vol 145 (6) ◽  
pp. 1277-1292 ◽  
Author(s):  
Stacy M. Stabler ◽  
Lisa L. Ostrowski ◽  
Susan M. Janicki ◽  
Mervyn J. Monteiro
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cell Death ◽  
Hela Cells ◽  
Calcium Binding ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
Cellular Functions ◽  
Disease States ◽  
Presenilin 2

It is well established that mutations in the presenilin 1 and 2 genes cause the majority of early onset Alzheimer's disease (AD). However, our understanding of the cellular functions of the proteins they encode remains rudimentary. Knowledge of proteins with which the presenilins interact should lead to a better understanding of presenilin function in normal and disease states. We report here the identification of a calcium-binding protein, calmyrin, that interacts preferentially with presenilin 2 (PS2). Calmyrin is myristoylated, membrane-associated, and colocalizes with PS2 when the two proteins are overexpressed in HeLa cells. Yeast two-hybrid liquid assays, affinity chromatography, and coimmunoprecipitation experiments confirm binding between PS2 and calmyrin. Functionally, calmyrin and PS2 increase cell death when cotransfected into HeLa cells. These results allude to several provocative possibilities for a dynamic role of calmyrin in signaling, cell death, and AD.

MSA-35: a protein identified by human autoantibodies that colocalizes with microtubules

1992 ◽  
Vol 70 (10-11) ◽  
pp. 1115-1122 ◽  
Author(s):  
J. B. Rattner ◽  
T. Wang ◽  
G. Mack ◽  
L. Martin ◽  
M. J. Fritzler
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Hela Cells ◽  
Temporal Distribution ◽  
Mitotic Arrest ◽  
Spatial And Temporal Distribution ◽  
Intercellular Bridge ◽  
Centrosome Separation ◽  
Interphase Cells ◽  
Low Levels

We have identified a putative 35-kilodalton protein that colocalizes with microtubules and displays a unique spatial and temporal distribution during the cell cycle of HeLa cells. This protein has been given the designation MSA-35. MSA-35 first appears in association with microtubules and centrosomes of interphase cells exhibiting centrosome separation as a prelude to cell division. This protein is found in conjunction with kinetochore microtubules throughout their appearance. MSA-35 transiently associates with interpolar microtubules following anaphase and the pattern of MSA-35 reactivity in telophase cells suggests that there are at least seven domains within the intercellular bridge. The distribution of MSA-35 during and following recovery from mitotic arrest with nocodazole suggest that it is also present at low levels in interphase cells, can associate with interphase centrosomes, and colocalizes with nascent microtubules. The complex spatial and temporal distribution of MSA-35 indicates that it may be necessary for a series of events in the mitotic process such as the bundling of microtubules.Key words: mitosis, autoantibodies, spindle.

scholarly journals Dynamic redistribution of calmodulin in HeLa cells during cell division as revealed by a GFP-calmodulin fusion protein technique

1999 ◽  
Vol 112 (10) ◽  
pp. 1567-1577 ◽  
Author(s):  
C.J. Li ◽  
R. Heim ◽  
P. Lu ◽  
Y. Pu ◽  
R.Y. Tsien ◽  
...  
Keyword(s):  
Cell Division ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Membrane Fraction ◽  
Fluorescent Protein ◽  
Active Role ◽  
Biochemical Properties ◽  
Equatorial Region ◽  
Cleavage Furrow ◽  
Inhibitory Peptide

It has been suggested by many studies that Ca2+ signaling plays an important role in regulating key steps in cell division. In order to study the down stream components of calcium signaling, we have fused the gene of calmodulin (CaM) with that of green fluorescent protein (GFP) and expressed it in HeLa cells. The GFP-CaM protein was found to have similar biochemical properties as the wild-type CaM, and its distribution was also similar to that of the endogenous CaM. Using this GFP-tagged CaM as a probe, we have conducted a detailed examination of the spatial- and temporal-dependent redistribution of calmodulin in living mammalian cells during cell division. Our major findings are: (1) high density of CaM was found to distribute in two sub-cellular locations during mitosis; one fraction was concentrated in the spindle poles, while the other was concentrated in the sub-membrane region around the cell. (2) The sub-membrane fraction of CaM became aggregated at the equatorial region where the cleavage furrow was about to form. The timing of this localized aggregation of CaM was closely associated with the onset of cytokinesis. (3) Using a TA-CaM probe, we found that the sub-membrane fraction of CaM near the cleavage furrow was selectively activated during cell division. (4) When we injected a CaM-specific inhibitory peptide into early anaphase cells, cytokinesis was either blocked or severely delayed. These findings suggest that, in addition to Ca2+ ion, CaM may represent a second signal that can also play an active role in determining the positioning and timing of the cleavage furrow formation.

scholarly journals PERSISTENCE OF MESSENGER RNA THROUGH MITOSIS IN HELA CELLS

1969 ◽  
Vol 40 (2) ◽  
pp. 497-507 ◽  
Author(s):  
L. D. Hodge ◽  
E. Robbins ◽  
M. D. Scharff
Keyword(s):  
Electron Microscopy ◽  
Protein Synthesis ◽  
Cell Division ◽  
Hela Cells ◽  
Messenger Rna ◽  
Mammalian Cells ◽  
Actinomycin D ◽  
Progressive Increase ◽  
The Reformation

The decrease in protein synthesis which occurs in mammalian cells during cell division is associated with significant disaggregation of polyribosomes. For determining whether messenger RNA survives this disaggregation, the reformation of polyribosomes was investigated in synchronized HeLa cells as they progressed from metaphase into interphase in the presence of 2 µg/ml Actinomycin D. The persistence of messenger during cell division was evidenced by: (1) a progressive increase in the rate of protein synthesis in both treated and untreated cells for 45 min after metaphase; (2) reformation of polyribosomes, as determined by both sucrose gradients and electron microscopy, within 30 min after the addition of Actinomycin D to metaphase cells; (3) the persistence of approximately 50% of the rapidly labeled nonribosomal RNA which had associated with polyribosomes just before metaphase; (4) the resumption of synthesis, following cell division, of 6 selected peptides in Actinomycin-treated cells.

scholarly journals Phosphoinositide Signaling Pathways in Nuclei Are Associated with Nuclear Speckles Containing Pre-mRNA Processing Factors

1998 ◽  
Vol 9 (12) ◽  
pp. 3547-3560 ◽  
Author(s):  
Igor V. Boronenkov ◽  
Joost C. Loijens ◽  
Masato Umeda ◽  
Richard A. Anderson
Keyword(s):  
Signaling Pathways ◽  
Mammalian Cells ◽  
Second Messengers ◽  
Mrna Processing ◽  
Type I ◽  
Nuclear Speckles ◽  
Phosphoinositide Signaling ◽  
Antibody Staining ◽  
Phosphatidylinositol 4 ◽  
Processing Factors

Phosphoinositide signal transduction pathways in nuclei use enzymes that are indistinguishable from their cytosolic analogues. We demonstrate that distinct phosphatidylinositol phosphate kinases (PIPKs), the type I and type II isoforms, are concentrated in nuclei of mammalian cells. The cytosolic and nuclear PIPKs display comparable activities toward the substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate. Indirect immunofluorescence revealed that these kinases were associated with distinct subnuclear domains, identified as “nuclear speckles,” which also contained pre-mRNA processing factors. A pool of nuclear phosphatidylinositol bisphosphate (PIP2), the product of these kinases, was also detected at these same sites by monoclonal antibody staining. The localization of PIPKs and PIP2 to speckles is dynamic in that both PIPKs and PIP2 reorganize along with other speckle components upon inhibition of mRNA transcription. Because PIPKs have roles in the production of most phosphatidylinositol second messengers, these findings demonstrate that phosphatidylinositol signaling pathways are localized at nuclear speckles. Surprisingly, the PIPKs and PIP2 are not associated with invaginations of the nuclear envelope or any nuclear membrane structure. The putative absence of membranes at these sites suggests novel mechanisms for the generation of phosphoinositides within these structures.

Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

1967 ◽  
Vol 25 ◽  
pp. 136-137
Author(s):  
J. P. Petrali ◽  
E. J. Donati ◽  
L. A. Sternberger
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Specific Antiserum ◽  
Electron Microscopic ◽  
E Coli ◽  
Cytoplasmic Membranes ◽  
Viral Progeny ◽  
Ultrathin Sections ◽  
Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

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