Abstract 210: αMyHC-mCherry-LC3 Transgenic Mice is a New and Useful Tool to Examine the Role of Autophagy in Cardiomyocytes

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Mai Terada ◽  
Kiyoshi Nobori ◽  
Yoshiko Munehisa ◽  
Manabu Kakizaki ◽  
Takayoshi Ohba ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Heart Diseases ◽  
Hydrolytic Enzymes ◽  
Smooth Muscles ◽  
Membrane Vesicles ◽  
Double Positive ◽  
Intracellular Process ◽  
Fluorescence Protein ◽  
And Function

Autophagy is an intracellular process in which proteins and organelles are transported in double-membrane vesicles called autophagosomes through the cytoplasm to lysosomes for degradation. The autophagosome acquires hydrolytic enzymes by fusing with the lysosome to generate an autolysosome. Constitutive autophagy in the heart under baseline conditions is a homeostatic mechanism for maintaining cardiomyocyte size and global cardiac structure and function. Upregulation of autophagy in various heart diseases, including cardiac hypertrophy and heart failure, is an adaptive response for protecting cells from hemodynamic stress. However, the detailed roles of autophagy in the heart remain unclear. LC3 is localized on the autophagosome membrane. Exogenously expressed GFP fused to LC3 (GFP-LC3) serves as an ideal molecular marker for autophagosome. Transgenic mice expressing GFP-LC3 (CAG-GFP-LC3) have been used to detect autophagy systemically. However, CAG-GFP-LC3 mice cannot distinguish autophagy-positive cardiomyocytes from other cells such as fibroblasts and smooth muscles in the heart and cannot detect autolysosome because GFP-LC3 loses fluorescence due to lysosomal acidic and degradative conditions. To resolve these problems, we have generated transgenic mice (αMyHC-mCherry-LC3) expressing mCherry fused to LC3 under the control of αmyosin heavy chain promoter instead of CAG promoter to detect autophagy only in cardiomyocytes. mCherry is an improved-monomeric red-fluorescence protein and does not lose fluorescence under acidic condition. Thus, αMyHC-mCherry-LC3 mice can detect not only autophagosome before fusion with lysosome but also autophagosome after fusion with lysosome. Moreover, we have crossed αMyHC-mCherry-LC3 mice with CAG-GFP-LC3 mice. Green signals showed autophagosome in non-cardiomyocytes. On the other hand, red signals showed autolysosome and double positive signals showed autophagosome in cardiacmyocytes. In conclusion, we have generated αMyHC-mCherry-LC3 mice to detect both autophagosome and autolysosome. The double transgenic mice cannot only detect autophagosome and autolysosome but also distinguish between them. This is an innovative method to examine the role of autophagy in cardiomyocytes.

scholarly journals Expression of an epidermal keratin protein in liver of transgenic mice causes structural and functional abnormalities.

1995 ◽  
Vol 128 (1) ◽  
pp. 157-169 ◽  
Author(s):  
K M Albers ◽  
F E Davis ◽  
T N Perrone ◽  
E Y Lee ◽  
Y Liu ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Cell Structure ◽  
Structure And Function ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Inflammatory Infiltration ◽  
Intermediate Filament Proteins ◽  
And Function

To examine the role of keratin intermediate filament proteins in cell structure and function, transgenic mice were isolated that express a modified form of the human K14 keratin protein in liver hepatocytes. A modified K14 cDNA (K14.P) sequence was linked downstream of the mouse transthyretin (TTR) gene promoter and enhancer elements to achieve targeted expression in hepatocytes. Hepatocytes expressing high levels of the transgene were found to have abnormal keratin filament networks as detected by indirect immunofluorescence using an antibody specific for the transgene product. Light and electron microscopic level histological analysis of isolated liver tissue showed in many cases degenerative changes that included inflammatory infiltration, ballooning degeneration, an increase in fat containing vacuoles, and glycogen accumulation. These changes were most evident in older mice over four months of age. No indication of typical Mallory body structures were identified at either the light or electron microscopic level. To evaluate secretory function in transgenic livers, bile acid secretion rates were measured in isolated perfused liver and found to be approximately twofold lower than aged-matched controls. These findings indicate that expression of an abnormal keratin in liver epithelial cells in the in vivo setting can alter the structure and function of a tissue and suggest a role of the keratin network in cellular secretion.

scholarly journals Lysosome Function in Cardiovascular Diseases

2021 ◽  
Vol 55 (3) ◽  
pp. 277-300
Keyword(s):  
Intracellular Signaling ◽  
Hydrolytic Enzymes ◽  
Vascular Diseases ◽  
Sphingolipid Metabolism ◽  
Cellular Behavior ◽  
Cellular Processes ◽  
Vascular Regulation ◽  
Large Numbers ◽  
And Function

The lysosome is a single ubiquitous membrane-enclosed intracellular organelle with an acidic pH present in all eukaryotic cells, which contains large numbers of hydrolytic enzymes with their maximal enzymatic activity at a low pH (pH ≤ 5) such as proteases, nucleases, and phosphatases that are able to degrade extracellular and intracellular components. It is well known that lysosomes act as a center for degradation and recycling of large numbers of macromolecules delivered by endocytosis, phagocytosis, and autophagy. Lysosomes are recognized as key organelles for cellular clearance and are involved in many cellular processes and maintain cellular homeostasis. Recently, it has been shown that lysosome function and its related pathways are of particular importance in vascular regulation and related diseases. In this review, we highlighted studies that have improved our understanding of the connection between lysosome function and vascular physiological and pathophysiological activities in arterial smooth muscle cells (SMCs) and endothelial cells (ECs). Sphingolipids-metabolizingenzymes in lysosomes play critical roles in intracellular signaling events that influence cellular behavior and function in SMCs and ECs. The focus of this review will be to define the mechanism by which the lysosome contributes to cardiovascular regulation and diseases. It is believed that exploring the role of lysosomal function and its sphingolipid metabolism in the initiation and progression of vascular disease and regulation may provide novel insights into the understanding of vascular pathobiology and helps develop more effective therapeutic strategies for vascular diseases.

Analysis of the role of AML1-ETO in leukemogenesis, using an inducible transgenic mouse model

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2108-2115 ◽  
Author(s):  
Kristina L. Rhoades ◽  
Christopher J. Hetherington ◽  
Nari Harakawa ◽  
Donald A. Yergeau ◽  
Liming Zhou ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Myeloid Leukemia ◽  
Transgenic Mouse Model ◽  
Double Positive ◽  
Genetic Changes ◽  
The Central Nervous System ◽  
Responsive Element ◽  
Definitive Hematopoiesis ◽  
Tetracycline Inducible System

Abstract As reported previously, AML1-ETO knock-in mice were generated to investigate the role of AML1-ETO in leukemogenesis and to mimic the progression of t(8;21) leukemia. These knock-in mice died in midgestation because of hemorrhaging in the central nervous system and a block of definitive hematopoiesis during embryogenesis. Therefore, they are not a good model system for the development of acute myeloid leukemia. Therefore, mice were generated in which the expression of AML1-ETO is under the control of a tetracycline-inducible system. Multiple lines of transgenic mice have been produced with the AML1-ETO complementary DNA controlled by a tetracycline-responsive element. In the absence of the antibiotic tetracycline, AML1-ETO is strongly expressed in the bone marrow of AML1-ETO and tet-controlled transcriptional activator double-positive transgenic mice. Furthermore, the addition of tetracycline reduces AML1-ETO expression in double-positive mice to nondetectable levels. Throughout the normal murine lifespan of 24 months, mice expressing AML1-ETO have not developed leukemia. In spite of this, abnormal maturation and proliferation of progenitor cells have been observed from these animals. These results demonstrate that AML1-ETO has a very restricted capacity to transform cells. Either the introduction of additional genetic changes or the expression of AML1-ETO at a particular stage of hematopoietic cell differentiation will be necessary to develop a model for studying the pathogenesis of t(8;21).

scholarly journals Specific overexpression of IL-7 in the intestinal mucosa: the role in intestinal intraepithelial lymphocyte development

2008 ◽  
Vol 294 (6) ◽  
pp. G1421-G1430 ◽  
Author(s):  
Hua Yang ◽  
Blair Madison ◽  
Deborah L. Gumucio ◽  
Daniel H. Teitelbaum
Keyword(s):  
T Cell ◽  
Mouse Model ◽  
Transgenic Mice ◽  
Transgenic Mouse ◽  
Critical Role ◽  
Transgenic Mouse Model ◽  
Intraepithelial Lymphocytes ◽  
Specific Expression ◽  
And Function

IL-7 plays a crucial role in controlling T cell development and homeostasis. Since IL-7 may be derived from extraintestinal sources, and exogenous IL-7 broadly affects lymphoid populations, the actions of epithelial cell (EC)-derived IL-7 are not fully understood. The effect of intestinal specific expression of IL-7 on intestinal mucosal lymphocytes was investigated by using an IL-7 transgenic mouse model. We generated an intestinal EC-specific overexpressing IL-7 transgenic mouse model (IL-7vill) and compared their phenotype and function to wild-type C57BL/6J mice. EC-derived IL-7 overexpression was found to be exclusively in the small and large intestine. Numbers and subtypes of mucosal lymphocytes, including intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL), significantly changed in IL-7vill mice. From a functional standpoint, IEL proliferation also significantly increased in IL-7vill mice. IEL cytokine expression significantly changed in both T cell receptor (TCR)-αβ+ and TCR-γδ+ IEL subpopulations, including a significant increase in IFN-γ and TNF-α as well as an increase in keratinocyte growth factor expression. EC expression of CD103 (integrin αEβ7), the ligand of E-cadherin, markedly upregulated and may account for a mechanism of the massive expansion of IEL in transgenic mice. Systemic lymphoid populations did not change in transgenic mice. IL-7 overexpression by intestinal EC significantly affected IEL phenotype and function. These results offer insight into the role of IL-7 in IEL development and suggest a critical role of EC-derived expression of IL-7 in the phenotype and function of IEL.

scholarly journals The Multifaceted Functions of Autophagy in Breast Cancer Development and Treatment

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1447
Author(s):  
Nicolas J. Niklaus ◽  
Igor Tokarchuk ◽  
Mara Zbinden ◽  
Anna M. Schläfli ◽  
Paola Maycotte ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Fate ◽  
Hydrolytic Enzymes ◽  
Membrane Vesicles ◽  
Building Blocks ◽  
Breast Cancer Patients ◽  
Intracellular Content ◽  
Long Term Survivors

Macroautophagy (herein referred to as autophagy) is a complex catabolic process characterized by the formation of double-membrane vesicles called autophagosomes. During this process, autophagosomes engulf and deliver their intracellular content to lysosomes, where they are degraded by hydrolytic enzymes. Thereby, autophagy provides energy and building blocks to maintain cellular homeostasis and represents a dynamic recycling mechanism. Importantly, the clearance of damaged organelles and aggregated molecules by autophagy in normal cells contributes to cancer prevention. Therefore, the dysfunction of autophagy has a major impact on the cell fate and can contribute to tumorigenesis. Breast cancer is the most common cancer in women and has the highest mortality rate among all cancers in women worldwide. Breast cancer patients often have a good short-term prognosis, but long-term survivors often experience aggressive recurrence. This phenomenon might be explained by the high heterogeneity of breast cancer tumors rendering mammary tumors difficult to target. This review focuses on the mechanisms of autophagy during breast carcinogenesis and sheds light on the role of autophagy in the traits of aggressive breast cancer cells such as migration, invasion, and therapeutic resistance.

Analysis of the role of AML1-ETO in leukemogenesis, using an inducible transgenic mouse model

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2108-2115 ◽  
Author(s):  
Kristina L. Rhoades ◽  
Christopher J. Hetherington ◽  
Nari Harakawa ◽  
Donald A. Yergeau ◽  
Liming Zhou ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Myeloid Leukemia ◽  
Transgenic Mouse Model ◽  
Double Positive ◽  
Genetic Changes ◽  
The Central Nervous System ◽  
Responsive Element ◽  
Definitive Hematopoiesis ◽  
Tetracycline Inducible System

As reported previously, AML1-ETO knock-in mice were generated to investigate the role of AML1-ETO in leukemogenesis and to mimic the progression of t(8;21) leukemia. These knock-in mice died in midgestation because of hemorrhaging in the central nervous system and a block of definitive hematopoiesis during embryogenesis. Therefore, they are not a good model system for the development of acute myeloid leukemia. Therefore, mice were generated in which the expression of AML1-ETO is under the control of a tetracycline-inducible system. Multiple lines of transgenic mice have been produced with the AML1-ETO complementary DNA controlled by a tetracycline-responsive element. In the absence of the antibiotic tetracycline, AML1-ETO is strongly expressed in the bone marrow of AML1-ETO and tet-controlled transcriptional activator double-positive transgenic mice. Furthermore, the addition of tetracycline reduces AML1-ETO expression in double-positive mice to nondetectable levels. Throughout the normal murine lifespan of 24 months, mice expressing AML1-ETO have not developed leukemia. In spite of this, abnormal maturation and proliferation of progenitor cells have been observed from these animals. These results demonstrate that AML1-ETO has a very restricted capacity to transform cells. Either the introduction of additional genetic changes or the expression of AML1-ETO at a particular stage of hematopoietic cell differentiation will be necessary to develop a model for studying the pathogenesis of t(8;21).

Scanning electron microscopic study of collagen fibrillogenesis and extracellular compartmentalization in the chick embryo tendon

1986 ◽  
Vol 44 ◽  
pp. 208-209
Author(s):  
Grace C.H. Yang
Keyword(s):  
Chick Embryo ◽  
Extracellular Space ◽  
Fibroblast Cell ◽  
Collagen Fibrils ◽  
Electron Microscopic ◽  
Voltage Electron ◽  
Scanning Electron Microscopic Study ◽  
Microscopic Studies ◽  
And Function

The size and organization of collagen fibrils in the extracellular matrix is an important determinant of tissue structure and function. The synthesis and deposition of collagen involves multiple steps which begin within the cell and continue in the extracellular space. High-voltage electron microscopic studies of the chick embryo cornea and tendon suggested that the extracellular space is compartmentalized by the fibroblasts for the regulation of collagen fibril, bundle, and tissue specific macroaggregate formation. The purpose of this study is to gather direct evidence regarding the association of the fibroblast cell surface with newly formed collagen fibrils, and to define the role of the fibroblast in the control and the precise positioning of collagen fibrils, bundles, and macroaggregates during chick tendon development.

Role of the Cilia Membrane in Control of Microtubule Sliding

1980 ◽  
Vol 38 ◽  
pp. 612-615
Author(s):  
Edna S. Kaneshiro
Keyword(s):  
Control Mechanism ◽  
Beat Frequency ◽  
Surface Membrane ◽  
Ciliary Membrane ◽  
Ciliary Beating ◽  
Ciliary Reversal ◽  
Voltage Dependent ◽  
Reversal Mechanism ◽  
And Function

It is currently believed that ciliary beating results from microtubule sliding which is restricted in regions to cause bending. Cilia beat can be modified to bring about changes in beat frequency, cessation of beat and reversal in beat direction. In ciliated protozoans these modifications which determine swimming behavior have been shown to be related to intracellular (intraciliary) Ca2+ concentrations. The Ca2+ levels are in turn governed by the surface ciliary membrane which exhibits increased Ca2+ conductance (permeability) in response to depolarization. Mutants with altered behaviors have been isolated. Pawn mutants fail to exhibit reversal of the effective stroke of ciliary beat and therefore cannot swim backward. They lack the increased inward Ca2+ current in response to depolarizing stimuli. Both normal and pawn Paramecium made leaky to Ca2+ by Triton extrac¬tion of the surface membrane exhibit backward swimming only in reactivating solutions containing greater than IO-6 M Ca2+ Thus in pawns the ciliary reversal mechanism itself is left operational and only the control mechanism at the membrane is affected. The topographic location of voltage-dependent Ca2+ channels has been identified as a component of the ciliary mem¬brane since the inward Ca2+ conductance response is eliminated by deciliation and the return of the response occurs during cilia regeneration. Since the ciliary membrane has been impli¬cated in the control of Ca2+ levels in the cilium and therefore is the site of at least one kind of control of microtubule sliding, we have focused our attention on understanding the structure and function of the membrane.

POPDC proteins and cardiac function

2019 ◽  
Vol 47 (5) ◽  
pp. 1393-1404 ◽  
Author(s):  
Thomas Brand
Keyword(s):  
Potential Role ◽  
Striated Muscle ◽  
Short Review ◽  
Effector Proteins ◽  
Muscle Disease ◽  
Disease Genes ◽  
Protein Protein Interaction ◽  
Interaction Partners ◽  
And Function

Abstract The Popeye domain-containing gene family encodes a novel class of cAMP effector proteins in striated muscle tissue. In this short review, we first introduce the protein family and discuss their structure and function with an emphasis on their role in cyclic AMP signalling. Another focus of this review is the recently discovered role of POPDC genes as striated muscle disease genes, which have been associated with cardiac arrhythmia and muscular dystrophy. The pathological phenotypes observed in patients will be compared with phenotypes present in null and knockin mutations in zebrafish and mouse. A number of protein–protein interaction partners have been discovered and the potential role of POPDC proteins to control the subcellular localization and function of these interacting proteins will be discussed. Finally, we outline several areas, where research is urgently needed.

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