scholarly journals Characterization of proteins immunologically related to brain microtubule-associated protein MAP-1B in non-neural cells

1989 ◽  
Vol 92 (4) ◽  
pp. 607-620
Author(s):  
J. Diaz-Nido ◽  
J. Avila
Keyword(s):  
Mitotic Spindle ◽  
Polyclonal Antibodies ◽  
Microtubule Assembly ◽  
Neural Cells ◽  
Microtubule Network ◽  
Cytoplasmic Microtubule ◽  
Protein Map ◽  
Dividing Cells

Brain microtubule-associated protein MAP-1 is composed of at least two polypeptides, MAP-1A and MAP-1B, which are among the main components of the neural cytoskeleton. Specific monoclonal and polyclonal antibodies against MAP-1B stain nuclei, mitotic spindles, centrosomes and the cytoplasmic microtubule network of different non-neural cells studied by immunofluorescence microscopy. It appears that these cells contain two proteins of 325K and 220K (K = 10(3) Mr), which are immunologically related to brain MAP-1B. The 325K protein, which is localized to the cytoplasmic microtubule network, the centrosome and the mitotic spindle, seems to be structurally related to the neural MAP-1B, as judged from their similar peptide maps and phosphorylation patterns. The 220K protein, which is localized to the nuclear matrix in interphase cells and to the mitotic spindle in dividing cells, has a proteolytic profile different from that of neural MAP-1B and is phosphorylated to a much lesser extent than the 325K protein. Both proteins bind tubulin in vitro, which suggests that they may participate in microtubule assembly in vivo; the 325K protein could perform such a role during the entire cell cycle, while the 220K protein could be implicated in the formation of the mitotic spindle.

scholarly journals A microtubule-associated protein antigen unique to mitotic spindle microtubules in PtK1 cells.

1983 ◽  
Vol 96 (2) ◽  
pp. 424-434 ◽  
Author(s):  
J G Izant ◽  
J A Weatherbee ◽  
J R McIntosh
Keyword(s):  
Mitotic Spindle ◽  
Microtubule Network ◽  
Mitotic Apparatus ◽  
Microtubule Associated Proteins ◽  
Immunoglobulin Preparations ◽  
Rapid Dissolution ◽  
Associated Proteins ◽  
Cultured Human Cells

Microtubule-associated proteins (MAPs) that copurify with tubulin through multiple cycles of in vitro assembly have been implicated as regulatory factors and effectors in the in vivo activity of microtubules. As an approach to the analysis of the functions of these molecules, a collection of lymphocyte hybridoma monoclonal antibodies has been generated using MAPs from HeLa cell microtubule protein as antigen. Two of the hybridoma clones secrete IgGs that bind to distinct sites on what appears to be a 200,000-dalton polypeptide. Both immunoglobulin preparations stain interphase and mitotic apparatus microtubules in cultured human cells. One of the clones (N-3B4.3.10) secretes antibody that reacts only with cells of human origin, while antibody from the other hybridoma (N-2B5.11.2) cross-reacts with BSC and PtK1 cells, but not with 3T3 cells. In PtK1 cells the N-2B5 antigen is associated with the microtubules of the mitotic apparatus, but there is no staining of the interphase microtubule array; rather, the antibody stains an ill-defined juxtanuclear structure. Further, neither antibody stains vinblastine crystals in either human or marsupial cells at any stage of the cell cycle. N-2B5 antibody microinjected into living PtK1 cells binds to the mitotic spindle, but does not cause a rapid dissolution of either mitotic or interphase microtubule structures. When injected before the onset of anaphase, however, the N-2B5 antibody inhibits proper chromosome partition in mitotic PtK1 cells. N-2B5 antibody injected into interphase cells causes a redistribution of MAP antigen onto the microtubule network.

The stathmin phosphoprotein family: intracellular localization and effects on the microtubule network

1998 ◽  
Vol 111 (22) ◽  
pp. 3333-3346 ◽  
Author(s):  
O. Gavet ◽  
S. Ozon ◽  
V. Manceau ◽  
S. Lawler ◽  
P. Curmi ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Intracellular Signaling ◽  
Splice Variants ◽  
Intracellular Localization ◽  
Phosphorylation Site ◽  
Dependent Manner ◽  
Microtubule Depolymerization ◽  
Microtubule Network

Stathmin is a small regulatory phosphoprotein integrating diverse intracellular signaling pathways. It is also the generic element of a protein family including the neural proteins SCG10, SCLIP, RB3 and its two splice variants RB3′ and RB3″. Stathmin itself was shown to interact in vitro with tubulin in a phosphorylation-dependent manner, sequestering free tubulin and hence promoting microtubule depolymerization. We investigated the intracellular distribution and tubulin depolymerizing activity in vivo of all known members of the stathmin family. Whereas stathmin is not associated with interphase microtubules in HeLa cells, a fraction of it is concentrated at the mitotic spindle. We generated antisera specific for stathmin phosphoforms, which allowed us to visualize the regulation of phosphorylation-dephosphorylation during the successive stages of mitosis, and the partial localization of stathmin phosphorylated on serine 16 at the mitotic spindle. Results from overexpression experiments of wild-type and novel phosphorylation site mutants of stathmin further suggest that it induces depolymerization of interphase and mitotic microtubules in its unphosphorylated state but is inactivated by phosphorylation in mitosis. Phosphorylation of mutants 16A25A and 38A63A on sites 38 and 63 or 16 and 25, respectively, was sufficient for the formation of a functional spindle, whereas mutant 16A25A38A63E retained a microtubule depolymerizing activity. Transient expression of each of the neural phosphoproteins of the stathmin family showed that they are at least partially associated to the Golgi apparatus and not to other major membrane compartments, probably through their different NH2-terminal domains, as described for SCG10. Most importantly, like stathmin and SCG10, overexpressed SCLIP, RB3 and RB3″ were able to depolymerize interphase microtubules. Altogether, our results demonstrate in vivo the functional conservation of the stathmin domain within each protein of the stathmin family, with a microtubule destabilizing activity most likely essential for their specific biological function(s).

The TOGp protein is a new human microtubule-associated protein homologous to the Xenopus XMAP215

1998 ◽  
Vol 111 (10) ◽  
pp. 1371-1383 ◽  
Author(s):  
S. Charrasse ◽  
M. Schroeder ◽  
C. Gauthier-Rouviere ◽  
F. Ango ◽  
L. Cassimeris ◽  
...  
Keyword(s):  
Sequence Comparison ◽  
Immunofluorescence Microscopy ◽  
Open Reading Frame ◽  
Microtubule Assembly ◽  
Human Cell Lines ◽  
Reading Frame ◽  
Protein Map ◽  
Dividing Cells ◽  
Perinuclear Cytoplasm

We have recently identified a 6,449 bp cDNA, termed colonic, hepatic tumor over-expressed gene (ch-TOG), that is highly expressed in human tumors and brain. Its single open reading frame encodes a putative 218,000 Da polypeptide, TOGp. Antibodies generated against a bacterially expressed TOGp fragment specifically recognize a 218, 000 Da polypeptide in two human cell lines and in brain. Immunofluorescence microscopy using affinity-purified TOGp antibodies revealed that the distribution of TOGp was dependent upon the cell cycle. During interphase, TOGp was found concentrated in the perinuclear cytoplasm, where it co-localized with ER markers. In contrast anti-TOGp antibodies stained centrosomes and spindles in mitotic cells. TOGp co-sedimented with taxol-stabilized microtubules in vitro. Moreover, a TOGp enriched fraction promotes microtubule assembly both in solution and from nucleation centers. Finally, sequence comparison and immunologic cross-reaction suggest that TOGp is homologous to XMAP215, a previously described microtubule associated protein (MAP) from Xenopus eggs. These results suggest that TOGp is a MAP and that TOGp/XMAP215 may be necessary for microtubules rearrangements and spindle assembly in rapidly dividing cells.

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

scholarly journals Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1288
Author(s):  
Wendy Dong ◽  
Boris Kantor
Keyword(s):  
Large Scale ◽  
Lentiviral Vector ◽  
Clinical Applications ◽  
Scale Production ◽  
Base Editing ◽  
Epigenome Editing ◽  
Vector Systems ◽  
Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

scholarly journals A novel dephosphorylation targeting chimera selectively promoting tau removal in tauopathies

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Jie Zheng ◽  
Na Tian ◽  
Fei Liu ◽  
Yidian Zhang ◽  
Jingfen Su ◽  
...  
Keyword(s):  
Neurodegenerative Disorders ◽  
Protein Phosphatase ◽  
High Efficiency ◽  
Microtubule Assembly ◽  
Hyperphosphorylated Tau ◽  
Phosphatase 2A ◽  
Dependent Learning ◽  
The Brain

AbstractIntraneuronal accumulation of hyperphosphorylated tau is a hallmark pathology shown in over twenty neurodegenerative disorders, collectively termed as tauopathies, including the most common Alzheimer’s disease (AD). Therefore, selectively removing or reducing hyperphosphorylated tau is promising for therapies of AD and other tauopathies. Here, we designed and synthesized a novel DEPhosphorylation TArgeting Chimera (DEPTAC) to specifically facilitate the binding of tau to Bα-subunit-containing protein phosphatase 2A (PP2A-Bα), the most active tau phosphatase in the brain. The DEPTAC exhibited high efficiency in dephosphorylating tau at multiple AD-associated sites and preventing tau accumulation both in vitro and in vivo. Further studies revealed that DEPTAC significantly improved microtubule assembly, neurite plasticity, and hippocampus-dependent learning and memory in transgenic mice with inducible overexpression of truncated and neurotoxic human tau N368. Our data provide a strategy for selective removal of the hyperphosphorylated tau, which sheds new light for the targeted therapy of AD and related-tauopathies.

scholarly journals Drosophila Stathmin: A Microtubule-destabilizing Factor Involved in Nervous System Formation

2002 ◽  
Vol 13 (2) ◽  
pp. 698-710 ◽  
Author(s):  
Sylvie Ozon ◽  
Antoine Guichet ◽  
Olivier Gavet ◽  
Siegfried Roth ◽  
André Sobel
Keyword(s):  
Nervous System ◽  
System Development ◽  
Nervous System Development ◽  
Microtubule Assembly ◽  
Nervous Systems ◽  
Germ Cell Migration ◽  
Numerous Cell ◽  
First Time

Stathmin is a ubiquitous regulatory phosphoprotein, the generic element of a family of neural phosphoproteins in vertebrates that possess the capacity to bind tubulin and interfere with microtubule dynamics. Although stathmin and the other proteins of the family have been associated with numerous cell regulations, their biological roles remain elusive, as in particular inactivation of the stathmin gene in the mouse resulted in no clear deleterious phenotype. We identified stathmin phosphoproteins inDrosophila, encoded by a unique gene sharing the intron/exon structure of the vertebrate stathmin andstathmin family genes. They interfere with microtubule assembly in vitro, and in vivo when expressed in HeLa cells. Drosophila stathmin expression is regulated during embryogenesis: it is high in the migrating germ cells and in the central and peripheral nervous systems, a pattern resembling that of mammalian stathmin. Furthermore, RNA interference inactivation ofDrosophila stathmin expression resulted in germ cell migration arrest at stage 14. It also induced important anomalies in nervous system development, such as loss of commissures and longitudinal connectives in the ventral cord, or abnormal chordotonal neuron organization. In conclusion, a single Drosophilagene encodes phosphoproteins homologous to the entire vertebrate stathmin family. We demonstrate for the first time their direct involvement in major biological processes such as development of the reproductive and nervous systems.

Directionality of microtubule assembly: an in vivo study with the ciliate Tetrahymena

1978 ◽  
Vol 33 (1) ◽  
pp. 227-234
Author(s):  
S.F. Ng
Keyword(s):  
Tetrahymena Thermophila ◽  
Recessive Gene ◽  
Microtubule Assembly ◽  
In Vivo Study ◽  
Temperature Sensitive ◽  
Sensitive Mutant ◽  
Posterior Portion ◽  
Temperature Sensitive Mutant

A temperature-sensitive mutant homozygous for the recessive gene molb in Tetrahymena thermophila offers opportunity for studying the direction of microtubule assembly in vivo. At 39 degrees C the mutant fails to divide properly; the 2 daughter animals remain attached and bend over each other. As revealed by protargol staining, the bending results in acute turning and breaking of some of the longitudinal microtubular bands close and parallel to the surface. Hence, 2 broken microtubular ends are available for study of the problem of directionality of microtubule assembly, by assessing which of the 2 ends regenerates. In most cases the posterior portion of the longitudinal microtubular band regenerates. The present study hence supports the conclusion based on in vitro observation in other systems that microtubule assembly is predominantly unidirectional.

The centriolar complex isolated from starfish spermatozoa

1981 ◽  
Vol 49 (1) ◽  
pp. 33-49 ◽  
Author(s):  
R. Kuriyama ◽  
H. Kanatani
Keyword(s):  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Microtubule Assembly ◽  
High Concentration ◽  
Microtubule Organization ◽  
Sucrose Density Gradient Centrifugation ◽  
The Difference ◽  
Distal Centriole

Centrioles from spermatozoa of the starfish, Asterina pectinifera, were isolated and partially purified by solubilization of chromatin followed by sucrose density-gradient centrifugation. The ultrastructure of the isolated centriolar complex was investigated in whole mount preparations by electron microscopy. The complex unit was composed of a pair of centrioles and a pericentriolar structure, which associated with the distal end of the distal centriole by 9 spoke-like satellites extending radially to a marginal ring. Each satellite bifurcated at a dense node forming 2 fan-like shapes with a periodic striated pattern. The tubular structure of the centrioles easily disintegrated, leaving the pericentriolar structure or axonemal microtubules intact. The distal centriole in a spermatozoon served as an initiating site for flagellar microtubule assembly; that is, a number of “9 + 2′ axonemal tubules were observed adhering just beneath the distal end of the basal body. In experiments in vitro, polymerization of microtubule proteins purified from porcine brain was initiated by the structure at the ends of both proximal and distal centrioles, but not from the satellites or the marginal ring. Also, few if any microtubules were formed from the sides of each centriole, even in the presence of a high concentration of exogenous tubulin. On the other hand, centrioles of spermatozoa, when they were in mature ooplasm, could initiate the formation of sperm asters by microtubules. Therefore, centrioles in spermatozoa seem to be able to initiate microtubules in a 2 ways. A possible explanation of the difference between the 2 types of microtubule organization in vivo, i.e. in the sperm cell itself and in the ooplasm, it discussed.

Control of dorsoventral pattern in vertebrate neural development: induction and polarizing properties of the floor plate

Development ◽  
1991 ◽  
Vol 113 (Supplement_2) ◽  
pp. 105-122 ◽  
Author(s):  
Marysia Placzek ◽  
Toshiya Yamada ◽  
Marc Tessier-Lavigne ◽  
Thomas Jessell ◽  
Jane Dodd
Keyword(s):  
Neural Tube ◽  
Neural Development ◽  
Cell Types ◽  
Floor Plate ◽  
Neural Cells ◽  
Dorsoventral Patterning ◽  
Cellular Mechanisms ◽  
Cell Position

Distinct classes of neural cells differentiate at specific locations within the embryonic vertebrate nervous system. To define the cellular mechanisms that control the identity and pattern of neural cells we have used a combination of functional assays and antigenic markers to examine the differentiation of cells in the developing spinal cord and hindbrain in vivo and in vitro. Our results suggest that a critical step in the dorsoventral patterning of the embryonic CNS is the differentiation of a specialized group of midline neural cells, termed the floor plate, in response to local inductive signals from the underlying notochord. The floor plate and notochord appear to control the pattern of cell types that appear along the dorsoventral axis of the neural tube. The fate of neuroepithelial cells in the ventral neural tube may be defined by cell position with respect to the ventral midline and controlled by polarizing signals that originate from the floor plate and notochord.

Sign in / Sign up

Export Citation Format

Share Document