Detyrosinated (Glu) microtubules are stabilized by an ATP-sensitive plus-end cap

Many cell types contain a subset of long-lived, ‘stable’ microtubules that differ from dynamic microtubules in that they are enriched in post-translationally detyrosinated tubulin (Glu-tubulin). Elevated Glu tubulin does not stabilize the microtubules and the mechanism for the stability of Glu microtubules is not known. We used detergent-extracted cell models to investigate the nature of Glu microtubule stability. In these cell models, Glu microtubules did not incorporate exogenously added tubulin subunits on their distal ends, while >70% of the bulk microtubules did. Ca(2+)-generated fragments of Glu microtubules incorporated tubulin, showing that Glu microtubule ends are capped. Consistent with this, Glu microtubules in cell models were resistant to dilution-induced breakdown. Known microtubule end-associated proteins (EB1, APC, p150(Glued) and vinculin focal adhesions) were not localized on Glu microtubule ends. ATP, but not nonhydrolyzable analogues, induced depolymerization of Glu microtubules in cell models. Timelapse and photobleaching studies showed that ATP triggered subunit loss from the plus end. ATP breakdown of Glu microtubules was inhibited by AMP-PNP and vanadate, but not by kinase or other inhibitors. Additional experiments showed that conventional kinesin or kif3 were not involved in Glu microtubule capping. We conclude that Glu microtubules are stabilized by a plus-end cap that includes an ATPase with properties similar to kinesins.

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Post-translational modifications and stabilization of microtubules regulate transport of viral factors during infections

Biochemical Society Transactions ◽

10.1042/bst20210017 ◽

2021 ◽

Vol 49 (4) ◽

pp. 1735-1748

Author(s):

Silvia Requena ◽

Francisco Sánchez-Madrid ◽

Noa B. Martín-Cófreces

Keyword(s):

Immune Responses ◽

Immune Cells ◽

Building Blocks ◽

Cell Types ◽

Post Translational Modifications ◽

Cellular Processes ◽

Associated Proteins ◽

The Stability ◽

Different Characteristics ◽

Β Tubulin

Tubulin post-translational modifications (PTMs) constitute a source of diversity for microtubule (MT) functions, in addition to the different isotypes of α and β-tubulin acting as building blocks of MTs. Also, MT-associated proteins (MAPs) confer different characteristics to MTs. The combination of all these factors regulates the stability of these structures that act as rails to transport organelles within the cell, facilitating the association of motor complexes. All these functions are involved in crucial cellular processes in most cell types, ranging from spindle formation in mitosis to the defense against incoming cellular threats during phagocytosis mediated by immune cells. The regulation of MT dynamics through tubulin PTMs has evolved to depend on many different factors that act in a complex orchestrated manner. These tightly regulated processes are particularly relevant during the induction of effective immune responses against pathogens. Viruses have proved not only to hijack MTs and MAPs in order to favor an efficient infection, but also to induce certain PTMs that improve their cellular spread and lead to secondary consequences of viral processes. In this review, we offer a perspective on relevant MT-related elements exploited by viruses.

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A talin-dependent LFA-1 focal zone is formed by rapidly migrating T lymphocytes

The Journal of Cell Biology ◽

10.1083/jcb.200412032 ◽

2005 ◽

Vol 170 (1) ◽

pp. 141-151 ◽

Cited By ~ 141

Author(s):

Andrew Smith ◽

Yolanda R. Carrasco ◽

Paula Stanley ◽

Nelly Kieffer ◽

Facundo D. Batista ◽

...

Keyword(s):

Endothelial Cells ◽

T Lymphocytes ◽

Focal Adhesions ◽

Cell Types ◽

Intercellular Adhesion Molecule ◽

Lymphocyte Function ◽

Intercellular Adhesion Molecule 1 ◽

The Stability ◽

Focal Zone ◽

Cell Zone

Cells such as fibroblasts and endothelial cells migrate through the coordinated responses of discrete integrin-containing focal adhesions and complexes. In contrast, little is known about the organization of integrins on the highly motile T lymphocyte. We have investigated the distribution, activity, and cytoskeletal linkage of the integrin lymphocyte function associated antigen-1 (LFA-1) on human T lymphocytes migrating on endothelial cells and on ligand intercellular adhesion molecule-1 (ICAM-1). The pattern of total LFA-1 varies from low expression in the lamellipodia to high expression in the uropod. However, high affinity, clustered LFA-1 is restricted to a mid-cell zone that remains stable over time and over a range of ICAM-1 densities. Talin is essential for the stability and formation of the LFA-1 zone. Disruption of the talin–integrin link leads to loss of zone integrity and a substantial decrease in speed of migration on ICAM-1. This adhesive structure, which differs from the previously described integrin-containing attachments displayed by many other cell types, we have termed the “focal zone.”

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CLytA-DAAO Chimeric Enzyme Bound to Magnetic Nanoparticles. A New Therapeutical Approach for Cancer Patients?

International Journal of Molecular Sciences ◽

10.3390/ijms22031477 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1477

Author(s):

María Fuentes-Baile ◽

Elizabeth Pérez-Valenciano ◽

Pilar García-Morales ◽

Camino de Juan Romero ◽

Daniel Bello-Gil ◽

...

Keyword(s):

Magnetic Nanoparticles ◽

Colorectal Carcinoma ◽

Acid Oxidase ◽

Oxygen Species ◽

Cell Models ◽

Amino Acid Oxidase ◽

Direct Addition ◽

Alginate Capsules ◽

Anti Cancer ◽

The Stability

D-amino acid oxidase (DAAO) is an enzyme that catalyzes the oxidation of D-amino acids generating H2O2. The enzymatic chimera formed by DAAO bound to the choline-binding domain of N-acetylmuramoyl-L-alanine amidase (CLytA) induces cytotoxicity in several pancreatic and colorectal carcinoma and glioblastoma cell models. In the current work, we determined whether the effect of CLytA-DAAO immobilized in magnetic nanoparticles, gold nanoparticles, and alginate capsules offered some advantages as compared to the free CLytA-DAAO. Results indicate that the immobilization of CLytA-DAAO in magnetic nanoparticles increases the stability of the enzyme, extending its time of action. Besides, we compared the effect induced by CLytA-DAAO with the direct addition of hydrogen peroxide, demonstrating that the progressive generation of reactive oxygen species by CLytA-DAAO is more effective in inducing cytotoxicity than the direct addition of H2O2. Furthermore, a pilot study has been initiated in biopsies obtained from pancreatic and colorectal carcinoma and glioblastoma patients to evaluate the expression of the main genes involved in resistance to CLytA-DAAO cytotoxicity. Based on our findings, we propose that CLytA-DAAO immobilized in magnetic nanoparticles could be effective in a high percentage of patients and, therefore, be used as an anti-cancer therapy for pancreatic and colorectal carcinoma and glioblastoma.

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Urine-Derived Epithelial Cells as Models for Genetic Kidney Diseases

Cells ◽

10.3390/cells10061413 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1413

Author(s):

Tjessa Bondue ◽

Fanny O. Arcolino ◽

Koenraad R. P. Veys ◽

Oyindamola C. Adebayo ◽

Elena Levtchenko ◽

...

Keyword(s):

Epithelial Cells ◽

Kidney Diseases ◽

Cell Types ◽

Cell Models ◽

Tubular Epithelial Cells ◽

Disease Mechanisms ◽

Proximal Tubular Epithelial Cells ◽

Proximal Tubular ◽

Disease Cell

Epithelial cells exfoliated in human urine can include cells anywhere from the urinary tract and kidneys; however, podocytes and proximal tubular epithelial cells (PTECs) are by far the most relevant cell types for the study of genetic kidney diseases. When maintained in vitro, they have been proven extremely valuable for discovering disease mechanisms and for the development of new therapies. Furthermore, cultured patient cells can individually represent their human sources and their specific variants for personalized medicine studies, which are recently gaining much interest. In this review, we summarize the methodology for establishing human podocyte and PTEC cell lines from urine and highlight their importance as kidney disease cell models. We explore the well-established and recent techniques of cell isolation, quantification, immortalization and characterization, and we describe their current and future applications.

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Automated analysis of filopodial length and spatially resolved protein concentration via adaptive shape tracking

Molecular Biology of the Cell ◽

10.1091/mbc.e16-06-0406 ◽

2016 ◽

Vol 27 (22) ◽

pp. 3616-3626 ◽

Cited By ~ 4

Author(s):

Tanumoy Saha ◽

Isabel Rathmann ◽

Abhiyan Viplav ◽

Sadhana Panzade ◽

Isabell Begemann ◽

...

Keyword(s):

Protein Concentration ◽

Growth Dynamics ◽

Automated Analysis ◽

Cell Types ◽

Control Mechanisms ◽

Spatially Resolved ◽

Novel Approach ◽

Associated Proteins

Filopodia are dynamic, actin-rich structures that transiently form on a variety of cell types. To understand the underlying control mechanisms requires precise monitoring of localization and concentration of individual regulatory and structural proteins as filopodia elongate and subsequently retract. Although several methods exist that analyze changes in filopodial shape, a software solution to reliably correlate growth dynamics with spatially resolved protein concentration along the filopodium independent of bending, lateral shift, or tilting is missing. Here we introduce a novel approach based on the convex-hull algorithm for parallel analysis of growth dynamics and relative spatiotemporal protein concentration along flexible filopodial protrusions. Detailed in silico tests using various geometries confirm that our technique accurately tracks growth dynamics and relative protein concentration along the filopodial length for a broad range of signal distributions. To validate our technique in living cells, we measure filopodial dynamics and quantify spatiotemporal localization of filopodia-associated proteins during the filopodial extension–retraction cycle in a variety of cell types in vitro and in vivo. Together these results show that the technique is suitable for simultaneous analysis of growth dynamics and spatiotemporal protein enrichment along filopodia. To allow readily application by other laboratories, we share source code and instructions for software handling.

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Artificial metaplasia of pigmented epithelium into retina in tadpoles and adult frogs

Development ◽

10.1242/dev.28.3.521 ◽

1972 ◽

Vol 28 (3) ◽

pp. 521-546

Author(s):

G. V. Lopashov ◽

Alla A. Sologub

Keyword(s):

Rana Temporaria ◽

Cell Types ◽

Control Mechanisms ◽

General Control ◽

Pigmented Epithelium ◽

The Stability

The present work was aimed at investigating the possibility and conditions necessary for the artificial transformation of one tissue into another. Experiments were carried out with the pigmented epithelium of the eye in tadpoles and adult frogs of Rana temporaria. Following the removal of the mesenchyme envelopes (or their exfoliation during the experiment), pigmented epithelium transformed into retina under the influence of retina from tadpoles of the same species. This phenomenon was observed both under the cultivation of a piece of retina in a sandwich of pigmented epithelium and the transplantation of pigmented epithelium layers into the eye cavity of tadpoles. Such transformation did not occur in the absence of retinal influence. Metaplasia requires the removal of the mesenchyme envelopes, the action of the retinal agent, as well as preservation of the integrity of the pigmented epithelium layer and subsequent proliferation of its cells. The character of general control mechanisms both maintaining the stability of cell types and leading to their transformation into other cell types is discussed.

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Identification of stable reference genes in differentiating human pluripotent stem cells

Physiological Genomics ◽

10.1152/physiolgenomics.00130.2014 ◽

2015 ◽

Vol 47 (6) ◽

pp. 232-239 ◽

Cited By ~ 11

Author(s):

Gustav Holmgren ◽

Nidal Ghosheh ◽

Xianmin Zeng ◽

Yalda Bogestål ◽

Peter Sartipy ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Gene Expression Data ◽

Pluripotent Stem Cells ◽

Reference Genes ◽

Cell Types ◽

Human Pluripotent Stem Cells ◽

Expression Data ◽

Large Variability ◽

The Stability

Reference genes, often referred to as housekeeping genes (HKGs), are frequently used to normalize gene expression data based on the assumption that they are expressed at a constant level in the cells. However, several studies have shown that there may be a large variability in the gene expression levels of HKGs in various cell types. In a previous study, employing human embryonic stem cells (hESCs) subjected to spontaneous differentiation, we observed that the expression of commonly used HKG varied to a degree that rendered them inappropriate to use as reference genes under those experimental settings. Here we present a substantially extended study of the HKG signature in human pluripotent stem cells (hPSC), including nine global gene expression datasets from both hESC and human induced pluripotent stem cells, obtained during directed differentiation toward endoderm-, mesoderm-, and ectoderm derivatives. Sets of stably expressed genes were compiled, and a handful of genes (e.g., EID2, ZNF324B, CAPN10, and RABEP2) were identified as generally applicable reference genes in hPSCs across all cell lines and experimental conditions. The stability in gene expression profiles was confirmed by reverse transcription quantitative PCR analysis. Taken together, the current results suggest that differentiating hPSCs have a distinct HKG signature, which in some aspects is different from somatic cell types, and underscore the necessity to validate the stability of reference genes under the actual experimental setup used. In addition, the novel putative HKGs identified in this study can preferentially be used for normalization of gene expression data obtained from differentiating hPSCs.

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The Putative RNA-Binding Protein Nrp1 Promotes the Loading of Kinesin-14/Klp2 to the Mitotic Spindle and Is Sequestered Into Heat-Induced Protein Aggregates in Fission Yeast

10.20944/preprints202103.0452.v1 ◽

2021 ◽

Author(s):

Masashi Yukawa ◽

Mitsuki Ohishi ◽

Yusuke Yamada ◽

Takashi Toda

Keyword(s):

Fission Yeast ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Protein Aggregates ◽

Associated Proteins ◽

Spindle Microtubules ◽

The Stability ◽

And Function ◽

Post Transcriptional Regulation

Cells form a bipolar spindle during mitosis to ensure accurate chromosome segregation. Proper spindle architecture is established by a set of kinesin motors and microtubule-associated proteins. In most eukaryotes, kinesin-5 motors are essential for this process, and genetic or chemical inhibition of their activity leads to the emergence of monopolar spindles and cell death. However, these deficiencies can be rescued by simultaneous inactivation of kinesin-14 motors, as they counteract kinesin-5. We conducted detailed genetic analyses in fission yeast to understand the mechanisms driving spindle assembly in the absence of kinesin-5. Here we show that deletion of the nrp1 gene, which encodes a putative RNA-binding protein with unknown function, can rescue temperature sensitivity caused by cut7-22, a fission yeast kinesin-5 mutant. Interestingly, kinesin-14/Klp2 levels on the spindles in the cut7 mutants were significantly reduced by the nrp1 deletion, although the total levels of Klp2 and the stability of spindle microtubules remained unaffected. Moreover, RNA-binding motifs of Nrp1 are essential for its cytoplasmic localization and function. We have also found that a portion of Nrp1 is spatially and functionally sequestered by chaperone-based protein aggregates upon mild heat stress and limits cell division at high temperatures. We propose that Nrp1 might be involved in post-transcriptional regulation through its RNA-binding ability to promote the loading of Klp2 on the spindle microtubules.

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Microtubule-based Endoplasmic Reticulum Motility in Xenopus laevis: Activation of Membrane-associated Kinesin during Development

Molecular Biology of the Cell ◽

10.1091/mbc.10.6.1909 ◽

1999 ◽

Vol 10 (6) ◽

pp. 1909-1922 ◽

Cited By ~ 71

Author(s):

Jon D. Lane ◽

Victoria J. Allan

Keyword(s):

Endoplasmic Reticulum ◽

Cell Types ◽

Cytoplasmic Dynein ◽

Culture Cell ◽

Tissue Culture Cell ◽

Differential Interference Contrast Microscopy ◽

Animal Cells ◽

Microtubule Motor ◽

Directed Motility ◽

Conventional Kinesin

The endoplasmic reticulum (ER) in animal cells uses microtubule motor proteins to adopt and maintain its extended, reticular organization. Although the orientation of microtubules in many somatic cell types predicts that the ER should move toward microtubule plus ends, motor-dependent ER motility reconstituted in extracts ofXenopus laevis eggs is exclusively a minus end-directed, cytoplasmic dynein-driven process. We have used Xenopusegg, embryo, and somatic Xenopus tissue culture cell (XTC) extracts to study ER motility during embryonic development inXenopus by video-enhanced differential interference contrast microscopy. Our results demonstrate that cytoplasmic dynein is the sole motor for microtubule-based ER motility throughout the early stages of development (up to at least the fifth embryonic interphase). When egg-derived ER membranes were incubated in somatic XTC cytosol, however, ER tubules moved in both directions along microtubules. Data from directionality assays suggest that plus end-directed ER tubule extensions contribute ∼19% of the total microtubule-based ER motility under these conditions. In XTC extracts, the rate of ER tubule extensions toward microtubule plus ends is lower (∼0.4 μm/s) than minus end-directed motility (∼1.3 μm/s), and plus end-directed motility is eliminated by a function-blocking anti-conventional kinesin heavy chain antibody (SUK4). In addition, we provide evidence that the initiation of plus end-directed ER motility in somatic cytosol is likely to occur via activation of membrane-associated kinesin.

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Stabilization of the p53 transformation-related protein in mouse fibrosarcoma cell lines: effects of protein sequence and intracellular environment

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3385-3392.1989 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3385-3392

Author(s):

O Halevy ◽

A Hall ◽

M Oren

Keyword(s):

Cell Lines ◽

Primary Structure ◽

P53 Protein ◽

Cell Types ◽

Stable Complex ◽

Related Protein ◽

Fibrosarcoma Cell ◽

Mouse Tumors ◽

Cellular Environment ◽

The Stability

The transformation-related protein p53 is normally very labile. The stability of p53 is significantly increased in a number of fibrosarcoma cell lines derived from mouse tumors induced by treatment with physical or chemical agents. In many instances, p53 stabilization is correlated with the ability to form a stable complex with the heat shock protein cognate hsc70. We describe a line in which p53 is very stable yet has no detectable interaction with hsc70. The inability to form such a complex probably resides in the primary structure of the endogenous p53, since introduction of other p53 variants into those cells resulted in the appearance of a p53-hsc70 complex. The factors affecting p53 stability were investigated by stable transfection experiments. The results indicated that the primary structure of the p53 protein is a major determinant of its turnover rate; different p53 variants were degraded at distinct and characteristic rates in a number of transformed cell types. However, at least one p53 variant was degraded differently in nontransformed BALB/c-3T3 than in transformed fibrosarcoma cells, demonstrating that the specific cellular environment can also affect the stability of p53.

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