Fluorescence Micro-Spectroscopy Assessment of the in Vitro Dimerization of BACE1-GFP Fusion Protein in Cultured Cells

AbstractAlpha-synuclein (aSyn) participates in synaptic vesicle trafficking and synaptic transmission, but its misfolding is also strongly implicated in Parkinson’s disease (PD) and other neurodegenerative disorders known as synucleinopathies where misfolded aSyn accumulates in different regions of the central and peripheral nervous systems. Although increased aSyn expression levels or altered aggregation propensities likely underlie familial PD with SNCA amplification or mutations, the majority of synucleinopathies arise sporadically, indicating that disease can develop under normal levels of wildtype aSyn. We report here the development and characterization of a mouse line expressing an aSyn-GFP fusion protein under the control of native Snca regulatory elements. Regional and subcellular localization of the aSyn-GFP fusion protein in brains and peripheral tissues of knock-in (KI) mice are indistinguishable from that of wildtype littermates. Importantly, similar to wildtype aSyn, aSyn-GFP disperses from synaptic vesicles upon membrane depolarization, indicating that the tag does not alter normal aSyn dynamics at synapses. In addition, intracerebral injection of aSyn pre-formed fibrils into KI mice induced the formation of aSyn-GFP inclusions with a distribution pattern similar to that observed in wildtype mice, albeit with attenuated kinetics due to the GFP tag. We anticipate that this new mouse model will facilitate in vitro and in vivo studies requiring in situ detection of endogenous aSyn, therefore providing new insights into aSyn function in health and disease.Significance StatementAlpha-synuclein (aSyn) participates in synaptic vesicle function and represents a major component of the Lewy pathology found in Parkinson’s and related neurodegenerative diseases. The function of aSyn and the sequence of events leading to its aggregation and neurotoxicity are not fully understood. Here we present a new mouse model in which Enhanced Green Fluorescence Protein (GFP) has been knocked-in at the C-terminal of the Snca gene. The resulting fusion protein shows identical expression and localization to that of wildtype animals, is functional, and is incorporated into pathological aggregates in vitro and in vivo. This new tool allows for monitoring aSyn under a variety of physiological and pathological conditions, and may uncover additional insights into its function and dysfunction.

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Single molecular assay of individual ATP turnover by a myosin-GFP fusion protein expressed in vitro

FEBS Letters ◽

10.1016/s0014-5793(97)00359-1 ◽

1997 ◽

Vol 407 (2) ◽

pp. 235-238 ◽

Cited By ~ 29

Author(s):

Atsuko Hikikoshi Iwane ◽

Takashi Funatsu ◽

Yoshie Harada ◽

Makio Tokunaga ◽

Osamu Ohara ◽

...

Keyword(s):

Fusion Protein ◽

Molecular Assay ◽

Atp Turnover ◽

Gfp Fusion Protein

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Cell-free expression of a GFP fusion protein allows quantitation in vitro and in vivo

Current Biology ◽

10.1016/s0960-9822(06)00100-x ◽

1997 ◽

Vol 7 (4) ◽

pp. R207-R208 ◽

Cited By ~ 12

Author(s):

Theodore W. Kahn ◽

Roger N. Beachy ◽

Matthias M. Falk

Keyword(s):

Fusion Protein ◽

Free Expression ◽

Gfp Fusion Protein

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Identification and Classification of LEA Family Genes in Orchids and Characterization of Their Role in Callus Formation

10.21203/rs.2.12901/v1 ◽

2019 ◽

Author(s):

Chunmei He ◽

Xuncheng Liu ◽

Jaime A. Teixeira da Silva ◽

Haobin Wang ◽

Tao Peng ◽

...

Keyword(s):

Fusion Protein ◽

Callus Formation ◽

Late Embryogenesis Abundant ◽

Positive Role ◽

Lea Genes ◽

Gfp Fusion Protein ◽

Genome Wide ◽

Comprehensive Information ◽

Transgenic Lines

Abstract The plant late embryogenesis abundant (LEA) proteins are abundant in seeds, play an important role in various abiotic stresses. However, there is still no information on genome-wide identification of LEA genes in orchids and their function in callus formation is almost unknown. In this study, the LEA genes from two orchids (Phalaenopsis equestris and Dendrobium officinale), were genome-wide identified, classified and characterized. A total of 57 and 59 LEA genes were identified in the genomes and these were divided into 8 and 9 groups for P. equestris and D. officinale, respectively. The LEA_1 and LEA_4 genes from P. equestris and D. officinale showed strong expression in seeds, but were significantly down-regulated in flowers and absent in vegetative organs (leaves, stems and roots). In addition, the LEA_1 and LEA_4 genes from D. officinale were abundant in the protocorm-like body (PLB) stage, while weak signals that were detected in in vitro shoots could not be detected in plantlets. The expression of these genes highlights PLBs in orchids are somatic embryos. The DoLEA36 from LEA_4 and DoLEA43 from LEA_1 were further characterized. The GFP signal of the DoLEA36-GFP fusion protein was only detected in the cytoplasm, while the GFP signal of the DoLEA43-GFP fusion protein was detected in both the cytoplasm and nucleus. This indicates that DoEA36 localizes in the cytoplasm while DoLEA43 localizes in both the cytoplasm and nucleus. Both DoLEA36 and DoLEA43 stimulated callus formation in transgenic Arabidopsis. The percentage of callus formation from 35S::DoLEA43 transgenic lines was higher than in wild type plants in two callus induction methods. Our results provide comprehensive information about the LEA gene family in orchids and genetic evidence for the involvement of LEA genes in the induction of callus, which may reveal their positive role in the maintenance of PLBs in orchids.

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Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor

Cytometry ◽

10.1002/(sici)1097-0320(19990401)35:4<353::aid-cyto8>3.0.co;2-m ◽

1999 ◽

Vol 35 (4) ◽

pp. 353-362 ◽

Cited By ~ 83

Author(s):

Roland Brock ◽

Irene H.L. Hamelers ◽

Thomas M. Jovin

Keyword(s):

Epidermal Growth Factor Receptor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Fusion Protein ◽

Cultured Cells ◽

Growth Factor Receptor ◽

Gfp Fusion Protein ◽

Epidermal Growth

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Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100053711 ◽

1982 ◽

Vol 40 ◽

pp. 210-211

Author(s):

M.J. Murphy ◽

R.R. Price ◽

J.C. Sloman

Keyword(s):

Cultured Cells ◽

Human Tumor ◽

Cell Type ◽

Tumor Mass ◽

Initial Cell ◽

Cloning Assay ◽

Human Tumor Cloning ◽

The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

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Preparation of cultured astrocytes for x-ray microanalysis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156924 ◽

1989 ◽

Vol 47 ◽

pp. 988-989

Author(s):

N.K.R. Smith ◽

K.E. Hunter ◽

P. Mobley ◽

L.P. Felpel

Keyword(s):

Cultured Cells ◽

Elemental Content ◽

Elemental Distribution ◽

Sprague Dawley ◽

X Ray ◽

Cultured Astrocytes ◽

Freeze Dried ◽

Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

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IN VITRO METHODS FOR THE STUDY OF THE ADENOHYPOPHYSIAL FUNCTIONS TO SECRETE GONADOTROPHIN

Acta Endocrinologica ◽

10.1530/acta.0.068s027 ◽

1971 ◽

Vol 68 (1_Suppl) ◽

pp. S27-S40 ◽

Cited By ~ 1

Author(s):

T. Kobayashi ◽

T. Kigawa ◽

M. Mizuno ◽

T. Watanabe

Keyword(s):

Cell Culture ◽

Organ Culture ◽

Cultured Cells ◽

Cell Sheet ◽

Short Term ◽

Hypothalamic Extract ◽

Numerous Cell ◽

Single Cell Culture ◽

Almost All

ABSTRACT There are several in vitro methods to analyse the function of the adenohypophysis or the mechanisms of its regulation. The present paper deals with single cell culture, organ culture and short term incubation techniques by which the morphology and gonadotrophin-secreting function of the adenohypophysis were studied. In trypsin-dispersed cell culture, the adenohypophysial cells showed extensive propagation to form numerous cell colonies and finally develop into a confluent monolayer cell sheet covering completely the surface of culture vessels. Almost all of the cultured cells, however, became chromophobic, at least at the end of the first week of cultivation, when gonadotrophin was detectable neither in the culture medium nor in the cells themselves. After the addition of the hypothalamic extract, gonadotrophin became detectable again, and basophilic or PAS-positive granules also reappeared within the cells, suggesting that the gonadotrophs were stimulated by the extract to produce gonadotrophin. In organ culture and short term incubation, the incorporation of [3H] leucine into the adenohypophysial cells in relation to the addition of hypothalamic extract was examined. It was obvious that the ability to incorporate [3H] leucine into the gonadotrophs in vitro was highly dependent upon the presence of the hypothalamic extract.

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