scholarly journals A microtubule-binding protein associated with membranes of the Golgi apparatus.

1986 ◽  
Vol 103 (6) ◽  
pp. 2229-2239 ◽  
Author(s):  
V J Allan ◽  
T E Kreis
Keyword(s):  
Golgi Apparatus ◽  
Culture Cell ◽  
Proteinase K ◽  
Microtubule Network ◽  
Microtubule Binding ◽  
Golgi Stack ◽  
Cell Extracts ◽  
Triton X

A monoclonal antibody (M3A5), raised against microtubule-associated protein 2 (MAP-2), recognized an antigen associated with the Golgi complex in a variety of non-neuronal tissue culture cells. In double immunofluorescence studies M3A5 staining was very similar to that of specific Golgi markers, even after disruption of the Golgi apparatus organization with monensin or nocodazole. M3A5 recognized one band of Mr approximately 110,000 in immunoblots of culture cell extracts; this protein, designated 110K, was enriched in Golgi stack fractions prepared from rat liver. The 110K protein has been shown to partition into the aqueous phase by Triton X-114 extraction of a Golgi-enriched fraction and was eluted after pH 11.0 carbonate washing. It is therefore likely to be a peripheral membrane protein. Proteinase K treatment of an isolated Golgi stack fraction resulted in complete digestion of the 110K protein, both in the presence and absence of Triton X-100. A the 110K protein is accessible to protease in intact vesicles in vitro, it is presumably located on the cytoplasmic face of the Golgi membrane in vivo. The 110K protein was able to interact specifically with taxol-polymerized microtubules in vitro. These results suggest that the 110K protein may serve to link the Golgi apparatus to the microtubule network and so may belong to a novel class of proteins: the microtubule-binding proteins.

scholarly journals Localization of BmpA on the Exposed Outer Membrane of Borrelia burgdorferi by Monospecific Anti-Recombinant BmpA Rabbit Antibodies

2004 ◽  
Vol 72 (4) ◽  
pp. 2280-2287 ◽  
Author(s):  
Junghee J. Shin ◽  
Anton V. Bryksin ◽  
Henry P. Godfrey ◽  
Felipe C. Cabello
Keyword(s):  
Borrelia Burgdorferi ◽  
Essential Role ◽  
Cross Reactivity ◽  
The Other ◽  
Proteinase K ◽  
Surface Localization ◽  
Triton X ◽  
Limited Surface

ABSTRACT BmpA (P39) is an immunodominant chromosomally encoded Borrelia burgdorferi protein. The potential strong cross-reactivity of anti-BmpA antibodies with the other members of this paralogous protein family and the previous use of antibodies whose reactivity to the other Bmp proteins was uncharacterized have resulted in continued controversy over its localization in B. burgdorferi. In an effort to provide a definitive demonstration of the localization of BmpA, rabbit antibodies raised to recombinant BmpA (rBmpA) were rendered monospecific by absorption with rBmpB. This reagent did not react with rBmpB, rBmpC, or rBmpD in dot immunobinding, detected only a single 39-kDa band and a single 39-kDa, pI 5.0 spot on one- and two-dimensional immunoblots of B. burgdorferi lysates, respectively, and immunoprecipitated a single 39-kDa protein from these lysates. It detected BmpA in the Triton X-114-soluble and -insoluble fractions of B. burgdorferi, suggesting association with both inner and outer bacterial cell membranes. Treatment of intact B. burgdorferi with proteinase K partially digested BmpA, consistent with a limited surface exposure on the outer bacterial membrane, a suggestion confirmed by immunofluorescence of unfixed B. burgdorferi cultured in vitro and in vivo. Anti-rBmpA antibody was bacteriostatic for B. burgdorferi B31 in culture, again suggesting localization of BmpA on the exposed spirochetal outer surface. Surface localization of BmpA, growth inhibition by anti-rBmpA antibodies, and the previously reported conservation of bmpA in different B. burgdorferi sensu lato strains may indicate that BmpA plays an essential role in B. burgdorferi biology.

Two splice variants of Golgi-microtubule-associated protein of 210 kDa (GMAP-210) differ in their binding to the cis-Golgi network

2001 ◽  
Vol 357 (3) ◽  
pp. 699-708 ◽  
Author(s):  
Francisco RAMOS-MORALES ◽  
Carmen VIME ◽  
Michel BORNENS ◽  
Concepción FEDRIANI ◽  
Rosa M. RIOS
Keyword(s):  
Golgi Apparatus ◽  
Structural Integrity ◽  
Fluorescent Protein ◽  
Genomic Sequence ◽  
Splice Variants ◽  
Microtubule Network ◽  
Terminal Domain ◽  
Golgi Network

GMAP-210 (Golgi-microtubule-associated protein of 210kDa) is a peripheral Golgi protein that interacts with the minus end of microtubules through its C-terminus and with cis-Golgi network membranes through its N-terminus; it participates in the maintenance of the structural integrity of the Golgi apparatus [Infante, Ramos-Morales, Fedriani, Bornens and Rios (1999) J. Cell Biol. 145, 83–98]. We report here the cloning of a new isoform of GMAP-210 that lacks amino acid residues 105–196. On the basis of the analysis of the gmap-210 genomic sequence, we propose that the small isoform, GMAP-200, arises from alternative splicing of exon 4 of the primary transcript. Overexpression of GMAP-200 induces perturbations in both the Golgi apparatus and the microtubule network that are similar to those previously reported for GMAP-210 overexpression. We show that both isoforms are able to oligomerize under overexpression conditions. Analysis in vitro and in vivo, with the green fluorescent protein as a marker, reveals that the binding of the N-terminal domain of GMAP-200 to the cis-Golgi network membranes is lower than that of the N-terminal domain of GMAP-210. Implications for the regulation of interaction between the cis-Golgi network and microtubules are discussed.

scholarly journals Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly

2018 ◽  
Vol 29 (6) ◽  
pp. 722-735 ◽  
Author(s):  
Sarah Bianchi ◽  
Kacper B. Rogala ◽  
Nicola J. Dynes ◽  
Manuel Hilbert ◽  
Sebastian A. Leidel ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Microtubule Network ◽  
Microtubule Binding ◽  
C Elegans ◽  
Evolutionarily Conserved ◽  
Organelle Assembly ◽  
In Vivo Analysis ◽  
Cilia And Flagella

Centrioles are microtubule-based organelles that organize the microtubule network and seed the formation of cilia and flagella. New centrioles assemble through a stepwise process dependent notably on the centriolar protein SAS-5 in Caenorhabditis elegans. SAS-5 and its functional homologues in other species form oligomers that bind the centriolar proteins SAS-6 and SAS-4, thereby forming an evolutionarily conserved structural core at the onset of organelle assembly. Here, we report a novel interaction of SAS-5 with microtubules. Microtubule binding requires SAS-5 oligomerization and a disordered protein segment that overlaps with the SAS-4 binding site. Combined in vitro and in vivo analysis of select mutants reveals that the SAS-5–microtubule interaction facilitates centriole assembly in C. elegans embryos. Our findings lead us to propose that the interdependence of SAS-5 oligomerization and microtubule binding reflects an avidity mechanism, which also strengthens SAS-5 associations with other centriole components and, thus, promotes organelle assembly.

scholarly journals GMAP-210, A Cis-Golgi Network-associated Protein, Is a Minus End Microtubule-binding Protein

1999 ◽  
Vol 145 (1) ◽  
pp. 83-98 ◽  
Author(s):  
Carlos Infante ◽  
Francisco Ramos-Morales ◽  
Concepción Fedriani ◽  
Michel Bornens ◽  
Rosa M. Rios
Keyword(s):  
Golgi Apparatus ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Coiled Coil ◽  
Microtubule Network ◽  
Microtubule Binding ◽  
Terminal Domain ◽  
Green Fluorescent ◽  
Golgi Network

We report that a peripheral Golgi protein with a molecular mass of 210 kD localized at the cis-Golgi network (Rios, R.M., A.M. Tassin, C. Celati, C. Antony, M.C. Boissier, J.C. Homberg, and M. Bornens. 1994. J. Cell Biol. 125:997–1013) is a microtubule-binding protein that associates in situ with a subpopulation of stable microtubules. Interaction of this protein, now called GMAP-210, for Golgi microtubule-associated protein 210, with microtubules in vitro is direct, tight and nucleotide-independent. Biochemical analysis further suggests that GMAP-210 specifically binds to microtubule ends. The full-length cDNA encoding GMAP-210 predicts a protein of 1,979 amino acids with a very long central coiled-coil domain. Deletion analyses in vitro show that the COOH terminus of GMAP-210 binds to microtubules whereas the NH2 terminus binds to Golgi membranes. Overexpression of GMAP-210–encoding cDNA induced a dramatic enlargement of the Golgi apparatus and perturbations in the microtubule network. These effects did not occur when a mutant lacking the COOH-terminal domain was expressed. When transfected in fusion with the green fluorescent protein, the NH2-terminal domain associated with the cis-Golgi network whereas the COOH-terminal microtubule-binding domain localized at the centrosome. Altogether these data support the view that GMAP-210 serves to link the cis-Golgi network to the minus ends of centrosome-nucleated microtubules. In addition, this interaction appears essential for ensuring the proper morphology and size of the Golgi apparatus.

Dementia with Lewy bodies—associated ß-synuclein mutations V70M and P123H cause mutation-specific neuropathological lesions

2021 ◽  
Author(s):  
Maryna Psol ◽  
Sofia Guerin Darvas ◽  
Kristian Leite ◽  
Sameehan U Mahajani ◽  
Mathias Bähr ◽  
...  
Keyword(s):  
Neuronal Network ◽  
Dementia With Lewy Bodies ◽  
Lewy Bodies ◽  
Sporadic Case ◽  
Network Activity ◽  
Proteinase K ◽  
Neuronal Network Activity ◽  
Distinct Disease

Abstract ß-Synuclein (ß-Syn) has long been considered to be an attenuator for the neuropathological effects caused by the Parkinson’s disease-related α-Synuclein (α-Syn) protein. However, recent studies demonstrated that overabundant ß-Syn can form aggregates and induce neurodegeneration in CNS neurons in vitro and in vivo, albeit at a slower pace as compared to α-Syn. Here we demonstrate that ß-Syn mutants V70M, detected in a sporadic case of Dementia with Lewy Bodies (DLB), and P123H, detected in a familial case of DLB, robustly aggravate the neurotoxic potential of ß-Syn. Intriguingly, the two mutations trigger mutually exclusive pathways. ß-Syn V70M enhances morphological mitochondrial deterioration and degeneration of dopaminergic and non-dopaminergic neurons, but has no influence on neuronal network activity. Conversely, ß-Syn P123H silences neuronal network activity, but does not aggravate neurodegeneration. ß-Syn WT, V70M and P123H formed proteinase K (PK) resistant intracellular fibrils within neurons, albeit with less stable C-termini as compared to α-Syn. Under cell free conditions, ß-Syn V70M demonstrated a much slower pace of fibril formation as compared to WT ß-Syn, and P123H fibrils present with a unique phenotype characterized by large numbers of short, truncated fibrils. Thus, it is possible that V70M and P123H cause structural alterations in ß-Syn, that are linked to their distinct neuropathological profiles. The extent of the lesions caused by these neuropathological profiles is almost identical to that of overabundant α-Syn, and thus likely to be directly involved into etiology of DLB. Over all, this study provides insights into distinct disease mechanisms caused by mutations of ß-Syn.

scholarly journals Adipose and Muscle Cell Co-Culture System: A Novel In Vitro Tool to Mimic the In Vivo Cellular Environment

Biology ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 6
Author(s):  
Palaniselvam Kuppusamy ◽  
Dahye Kim ◽  
Ilavenil Soundharrajan ◽  
Inho Hwang ◽  
Ki Choon Choi
Keyword(s):  
Drug Development ◽  
Culture System ◽  
Cell Types ◽  
Culture Cell ◽  
In Vitro Techniques ◽  
Culture Systems ◽  
Complex Interactions ◽  
Cellular Environment

A co-culture system allows researchers to investigate the complex interactions between two cell types under various environments, such as those that promote differentiation and growth as well as those that mimic healthy and diseased states, in vitro. In this paper, we review the most common co-culture systems for myocytes and adipocytes. The in vitro techniques mimic the in vivo environment and are used to investigate the causal relationships between different cell lines. Here, we briefly discuss mono-culture and co-culture cell systems and their applicability to the study of communication between two or more cell types, including adipocytes and myocytes. Also, we provide details about the different types of co-culture systems and their applicability to the study of metabolic disease, drug development, and the role of secretory factors in cell signaling cascades. Therefore, this review provides details about the co-culture systems used to study the complex interactions between adipose and muscle cells in various environments, such as those that promote cell differentiation and growth and those used for drug development.

scholarly journals A complex control region of the mouse rRNA gene directs accurate initiation by RNA polymerase I.

1985 ◽  
Vol 5 (3) ◽  
pp. 554-562 ◽  
Author(s):  
K G Miller ◽  
J Tower ◽  
B Sollner-Webb
Keyword(s):  
Initiation Site ◽  
Rrna Gene ◽  
Sufficient Information ◽  
Reaction Conditions ◽  
Cell Extracts ◽  
S 100 ◽  
Initiation Reaction ◽  
Polymerase I

To determine the size and location of the mouse rDNA promoter, we constructed systematic series of deletion mutants approaching the initiation site from the 5' and 3' directions. These templates were transcribed in vitro under various conditions with S-100 and whole-cell extracts. Surprisingly, the size of the rDNA region that determines the level of transcription differed markedly, depending on the reaction conditions. In both kinds of cell extracts, the apparent 5' border of the promoter was at residue ca. -27 under optimal transcription conditions, but as reaction conditions became less favorable, the 5' border moved progressively out to residues -35, -39, and -45. The complete promoter, however, extends considerably further, for under other nonoptimal conditions, we observed major effects of promoter domains extending in the 5' direction to positions ca. -100 and -140. In contrast, the apparent 3' border of the mouse rDNA promoter was at residue ca. +9 under all conditions examined. We also show that the subcloned rDNA region from -39 to +9 contains sufficient information to initiate accurately and that the region between +2 and +9 can influence the specificity of initiation. These data indicate that, although the polymerase I transcription factors recognize and accurately initiate with only the sequences downstream of residue -40, sequences extending out to residue -140 greatly favor the initiation reaction; presumably, this entire region is involved in rRNA transcription in vivo.

scholarly journals Structural Variability of the Herpes Simplex Virus 1 GenomeIn VitroandIn Vivo

2012 ◽  
Vol 86 (16) ◽  
pp. 8592-8601 ◽  
Author(s):  
Charlotte Mahiet ◽  
Ayla Ergani ◽  
Nicolas Huot ◽  
Nicolas Alende ◽  
Ahmed Azough ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Considerable Proportion ◽  
Inverted Repeats ◽  
Herpes Simplex Virus 1 ◽  
Cell Extracts ◽  
Simplex Virus ◽  
Hsv 1

Herpes simplex virus 1 (HSV-1) is a human pathogen that leads to recurrent facial-oral lesions. Its 152-kb genome is organized in two covalently linked segments, each composed of a unique sequence flanked by inverted repeats. Replication of the HSV-1 genome produces concatemeric molecules in which homologous recombination events occur between the inverted repeats. This mechanism leads to four genome isomers (termed P, IS, IL, and ILS) that differ in the relative orientations of their unique fragments. Molecular combing analysis was performed on DNA extracted from viral particles and BSR, Vero, COS-7, and Neuro-2a cells infected with either strain SC16 or KOS of HSV-1, as well as from tissues of experimentally infected mice. Using fluorescence hybridization, isomers were repeatedly detected and distinguished and were accompanied by a large proportion of noncanonical forms (40%). In both cell and viral-particle extracts, the distributions of the four isomers were statistically equivalent, except for strain KOS grown in Vero and Neuro-2a cells, in which P and IS isomers were significantly overrepresented. In infected cell extracts, concatemeric molecules as long as 10 genome equivalents were detected, among which, strikingly, the isomer distributions were equivalent, suggesting that any such imbalance may occur during encapsidation.In vivo, for strain KOS-infected trigeminal ganglia, an unbalanced distribution distinct from the onein vitrowas observed, along with a considerable proportion of noncanonical assortment.

Characterization of an in vitro assay for import of 3-phosphoglycerate kinase into the glycosomes of Trypanosoma brucei

1990 ◽  
Vol 10 (9) ◽  
pp. 4545-4554
Author(s):  
J M Sommer ◽  
J A Thissen ◽  
M Parsons ◽  
C C Wang
Keyword(s):  
Trypanosoma Brucei ◽  
Protein Import ◽  
Sodium Dodecyl ◽  
Phosphoglycerate Kinase ◽  
Proteinase K ◽  
Vitro Assay ◽  
Protein Core ◽  
Triton X ◽  
Gamma S

Glycosomes are microbody organelles found in kinetoplastida, where they serve to compartmentalize the enzymes of the glycolytic pathway. In order to identify the mechanism by which these enzymes are targeted to the glycosome, we have modified the in vitro import assay developed by Dovey et al. (Proc. Natl. Acad. Sci. USA 85:2598-2602, 1988). This assay measures the uptake of in vitro-translated Trypanosoma brucei glycosomal 3-phosphoglycerate kinase (gPGK) by purified glycosomes. Up to 50% of the total 35S-gPGK in the glycosomal fraction was resistant to extraction by 3 M urea or treatment with proteinase K (500 micrograms/ml). The glycosome-associated 35S-gPGK could be chemically cross-linked to the endogenous glycosomal proteins to form a sodium dodecyl sulfate-resistant complex, suggesting that it is close to the intraglycosomal protein matrix. Deoxycholate solubilized the glycosome and thereby rendered the glycosome-associated 35S-gPGK fully susceptible to proteinase K. However, the glycosome-associated 35S-gPGK was not digested by proteinase K in the presence of Triton X-100, which cannot dissolve the glycosomal protein core. The 35S-gPGK synthesized in vitro was able to bind directly to protein cores, where it became resistant to urea extraction and proteinase K digestion. However, the 35S-gPGK-protein core complex exhibited a much higher density than the 35S-gPGK-glycosome complex and was readily separable in sucrose gradients. Thus, in our in vitro import assay, the 35S-gPGK appeared to associate with intact glycosomes, possibly reflecting import of protein into the organelle. Complete denaturation of the 35S-gPGK in 8 M urea prior to the assay enhanced the efficiency of its association with glycosomes. Native gPGK did not compete with the association of in vitro-translated gPGK unless it was denatured. The assay exhibited time and temperature dependence, but it did not require externally added ATP and was not inhibited by the nonhydrolyzable analogs adenosine-5'-(beta,gamma-imido)-triphosphate and gamma-S-ATP. However, the presence of 20 to 30 microM ATP inside the glycosome may fulfill the requirement for protein import.

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro

1987 ◽  
Vol 7 (10) ◽  
pp. 3694-3704
Author(s):  
C Prives ◽  
Y Murakami ◽  
F G Kern ◽  
W Folk ◽  
C Basilico ◽  
...  
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Early Gene ◽  
Wild Type ◽  
The Core ◽  
Cell Extracts ◽  
Sequence Requirements

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.

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