scholarly journals Cdc42 and formin activity control non-muscle myosin dynamics during Drosophila heart morphogenesis

2014 ◽  
Vol 206 (7) ◽  
pp. 909-922 ◽  
Author(s):  
Georg Vogler ◽  
Jiandong Liu ◽  
Timothy W. Iafe ◽  
Ede Migh ◽  
József Mihály ◽  
...  
Keyword(s):  
Cell Fate ◽  
Matrix Protein ◽  
Small Gtpase ◽  
Extracellular Matrix Protein ◽  
Specific Expression ◽  
Heart Morphogenesis ◽  
Lumen Formation ◽  
Spatiotemporal Regulation ◽  
Genetic Mechanisms ◽  
Heart Formation

During heart formation, a network of transcription factors and signaling pathways guide cardiac cell fate and differentiation, but the genetic mechanisms orchestrating heart assembly and lumen formation remain unclear. Here, we show that the small GTPase Cdc42 is essential for Drosophila melanogaster heart morphogenesis and lumen formation. Cdc42 genetically interacts with the cardiogenic transcription factor tinman; with dDAAM which belongs to the family of actin organizing formins; and with zipper, which encodes nonmuscle myosin II. Zipper is required for heart lumen formation, and its spatiotemporal activity at the prospective luminal surface is controlled by Cdc42. Heart-specific expression of activated Cdc42, or the regulatory formins dDAAM and Diaphanous caused mislocalization of Zipper and induced ectopic heart lumina, as characterized by luminal markers such as the extracellular matrix protein Slit. Placement of Slit at the lumen surface depends on Cdc42 and formin function. Thus, Cdc42 and formins play pivotal roles in heart lumen formation through the spatiotemporal regulation of the actomyosin network.

scholarly journals Regulation of neurogenesis and gliogenesis by the matricellular protein CCN2 in the mouse retina

2021 ◽  
Author(s):  
Golam Mohiuddin ◽  
Genesis Lopez ◽  
Jose Sinon ◽  
M. Elizabeth Hartnett ◽  
Anastasiia Bulakhova ◽  
...  
Keyword(s):  
Glial Cell ◽  
Cell Fate ◽  
Matrix Protein ◽  
Ex Vivo ◽  
Mouse Genetics ◽  
Extracellular Matrix Protein ◽  
Retinal Cell ◽  
Mouse Retina ◽  
Matricellular Protein ◽  
Retinal Progenitor

AbstractCellular communication network (CCN) 2 is an extracellular matrix protein with cell type- and context-dependent functions. Using a combination of mouse genetics and omic approaches, we show that CCN2 is expressed in early embryonic retinal progenitor cells (RPCs) and becomes restricted to fully differentiated Müller glial cells (MGCs) thereafter. Germline deletion of CCN2 in mice decreases BrdU labeling, reduces RPC pool, and impairs the competency of remaining RPCs to generate early and late born retinal cell types. Retinal hypocellularity and microphthalmia ensue. The transcriptomic changes associated with CCN2 inactivation include reduced marker and transcriptional regulator genes of retinal ganglion cells, photoreceptors and MGCs. Yap (Yes-associated protein), a singular node for transcriptional regulation of growth and differentiation genes, is also a target of CCN2 signals. In an organotypic model of ex vivo cultured embryonic retinas, CCN2 and YAP immunoreactivity signals overlap. Lentivirus-mediated YAP expression in CCN2-deficient retinal explants increases the number of differentiating Sox9-positive MGCs. Taken together, our data indicate that CCN2 controls the proliferative and differentiation potentials of RPCs ultimately endowing, a subpopulation thereof, with Müller glial cell fate.Summary statementA CCN2-YAP regulatory axis controls retinal progenitor cell growth and lineage commitment to neuronal and glial cell fates.

scholarly journals Multiple negative elements in a gene that codes for an extracellular matrix protein, collagen X, restrict expression to hypertrophic chondrocytes.

1993 ◽  
Vol 121 (5) ◽  
pp. 1173-1179 ◽  
Author(s):  
P Lu Valle ◽  
M Iwamoto ◽  
P Fanning ◽  
M Pacifici ◽  
B R Olsen
Keyword(s):  
Matrix Protein ◽  
Skeletal Development ◽  
Primary Cultures ◽  
Gene Promoter ◽  
Cell Types ◽  
Regulatory Elements ◽  
Hypertrophic Chondrocytes ◽  
Extracellular Matrix Protein ◽  
Specific Expression ◽  
X Gene

During skeletal development, chondrocytes go through several stages of differentiation. The last stage, chondrocyte hypertrophy, occurs in areas of endochondral ossification. Mature hypertrophic chondrocytes differ from immature chondrocytes in that they become postmitotic, increase their cellular volume up to eightfold, and synthesize a unique set of matrix molecules. One such molecule is a short collagenous protein, collagen X. Previous studies have shown that collagen X is not expressed by other cell types and that its specific expression in hypertrophic chondrocytes is controlled by transcriptional mechanisms. To define these mechanisms, plasmid constructs containing the chicken collagen X gene promoter and 5' flanking regions fused to a reporter gene (chloramphenicol acetyl transferase, CAT) were transfected into primary cultures of collagen X-expressing and nonexpressing cells. A construct containing a short (558 bp) promoter exhibited high levels of CAT activity in all cell types (fibroblasts, immature, and hypertrophic chondrocytes). Adding a 4.2-kb fragment of 5' flanking DNA to this construct resulted in a dramatic reduction of CAT activity in fibroblasts and immature chondrocytes, but had no effect in hypertrophic chondrocytes. Addition of three subfragments of the 4.2-kb fragment to the initial construct, either individually or in various combinations, showed that all subfragments reduced CAT activity somewhat in non-collagen X-expressing cells, and that their effects were additive. Unrelated DNA had no effect on CAT activity. The results suggest that multiple, diffuse upstream negative regulatory elements act in an additive manner to restrict transcription of the collagen X gene to hypertrophic chondrocytes.

scholarly journals Developmental Regulation of FKBP65

2000 ◽  
Vol 11 (11) ◽  
pp. 3925-3935 ◽  
Author(s):  
Charles E. Patterson ◽  
Theresa Schaub ◽  
Elaine J. Coleman ◽  
Elaine C. Davis
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Subcellular Fractionation ◽  
Immunohistochemical Localization ◽  
Extracellular Matrix Protein ◽  
Elastic Fibers ◽  
Airway Smooth Muscle Cells ◽  
Specific Expression ◽  
Adult Tissues ◽  
Mouse Tissues

FKBP65 (65-kDa FK506-binding protein) is a member of the highly conserved family of intracellular receptors called immunophilins. All have the property of peptidyl-prolyl cis-trans isomerization, and most have been implicated in folding and trafficking events. In an earlier study, we identified that FKBP65 associates with the extracellular matrix protein tropoelastin during its transport through the cell. In the present study, we have carried out a detailed investigation of the subcellular localization of FKBP65 and its relationship to tropoelastin. Using subcellular fractionation, Triton X-114 phase separation, protease protection assays, and immunofluorescence microscopy (IF), we have identified that FKBP65 is contained within the lumen of the endoplasmic reticulum (ER). Subsequent IF studies colocalized FKBP65 with tropoelastin and showed that the two proteins dissociate before reaching the Golgi apparatus. Immunohistochemical localization of FKBP65 in developing lung showed strong staining of vascular and airway smooth muscle cells. Similar areas stained positive for the presence of elastic fibers in the extracellular matrix. The expression of FKBP65 was investigated during development as tropoelastin is not expressed in adult tissues. Tissue-specific expression of FKBP65 was observed in 12-d old mouse tissues; however, the pattern of expression of FKBP65 was not restricted to those tissues expressing tropoelastin. This suggests that additional ligands for FKBP65 likely exist within the ER. Remarkably, in the adult tissues examined, FKBP65 expression was absent or barely detectable. Taken together, these results support an ER-localized FKBP65-tropoelastin interaction that occurs specifically during growth and development of tissues.

1280: Increased Susceptibility to Genitovaginal Prolapse Associated with a Polymorphism in the Promoter of the Extracellular Matrix Protein LAMC1

2007 ◽  
Vol 177 (4S) ◽  
pp. 421-422
Author(s):  
Ganka Nikolova ◽  
Christian O. Twiss ◽  
Hane Lee ◽  
Nelson Stanley ◽  
Janet Sinsheimer ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Extracellular Matrix Protein ◽  
Increased Susceptibility

scholarly journals Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor

2021 ◽  
Author(s):  
Aniel Moya-Torres ◽  
Monika Gupta ◽  
Fabian Heide ◽  
Natalie Krahn ◽  
Scott Legare ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Hollow Fiber ◽  
Mammalian Cells ◽  
Matrix Protein ◽  
Continuous Production ◽  
Extracellular Matrix Protein ◽  
Close Monitoring ◽  
High Quality ◽  
Biolayer Interferometry ◽  
Hollow Fiber Bioreactor

Abstract The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems. Key points • Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1 • Biolayer interferometry allows target protein quantitation in expression media • High production yields in the bioreactor do not impair Netrin-1 proteoglycan quality Graphical abstract

Direct interaction of the extracellular matrix protein DANCE with apolipoprotein(a) mediated by the kringle IV-type 2 domain

2002 ◽  
Vol 267 (4) ◽  
pp. 440-446 ◽  
Author(s):  
A. Kapetanopoulos ◽  
F. Fresser ◽  
G. Millonig ◽  
Y. Shaul ◽  
G. Baier ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Direct Interaction ◽  
Extracellular Matrix Protein ◽  
Apolipoprotein A

scholarly journals IκBα targeting promotes oxidative stress-dependent cell death

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Giovanna Carrà ◽  
Giuseppe Ermondi ◽  
Chiara Riganti ◽  
Luisella Righi ◽  
Giulia Caron ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Fate ◽  
Protein Interactions ◽  
In Silico ◽  
Inverse Correlation ◽  
Ros Production ◽  
Gene Encoding ◽  
Specific Expression ◽  
Novel Approach ◽  
Stress Dependent

Abstract Background Oxidative stress is a hallmark of many cancers. The increment in reactive oxygen species (ROS), resulting from an increased mitochondrial respiration, is the major cause of oxidative stress. Cell fate is known to be intricately linked to the amount of ROS produced. The direct generation of ROS is also one of the mechanisms exploited by common anticancer therapies, such as chemotherapy. Methods We assessed the role of NFKBIA with various approaches, including in silico analyses, RNA-silencing and xenotransplantation. Western blot analyses, immunohistochemistry and RT-qPCR were used to detect the expression of specific proteins and genes. Immunoprecipitation and pull-down experiments were used to evaluate protein-protein interactions. Results Here, by using an in silico approach, following the identification of NFKBIA (the gene encoding IκBα) amplification in various cancers, we described an inverse correlation between IκBα, oxidative metabolism, and ROS production in lung cancer. Furthermore, we showed that novel IκBα targeting compounds combined with cisplatin treatment promote an increase in ROS beyond the tolerated threshold, thus causing death by oxytosis. Conclusions NFKBIA amplification and IκBα overexpression identify a unique cancer subtype associated with specific expression profile and metabolic signatures. Through p65-NFKB regulation, IκBα overexpression favors metabolic rewiring of cancer cells and distinct susceptibility to cisplatin. Lastly, we have developed a novel approach to disrupt IκBα/p65 interaction, restoring p65-mediated apoptotic responses to cisplatin due to mitochondria deregulation and ROS-production.

scholarly journals Specific heparan sulfate modifications stabilize the synaptic organizer MADD-4/Punctin at C. elegans neuromuscular junctions

Genetics ◽  
2021 ◽  
Author(s):  
Mélissa Cizeron ◽  
Laure Granger ◽  
Hannes E BÜlow ◽  
Jean-Louis Bessereau
Keyword(s):  
Heparan Sulfate ◽  
Matrix Protein ◽  
Neuromuscular Junctions ◽  
Core Protein ◽  
Extracellular Matrix Protein ◽  
Inhibitory Synapses ◽  
Single Chain ◽  
C Elegans ◽  
Sugar Chains

Abstract Heparan sulfate proteoglycans contribute to the structural organization of various neurochemical synapses. Depending on the system, their role involves either the core protein or the glycosaminoglycan chains. These linear sugar chains are extensively modified by heparan sulfate modification enzymes, resulting in highly diverse molecules. Specific modifications of glycosaminoglycan chains may thus contribute to a sugar code involved in synapse specificity. Caenorhabditis elegans is particularly useful to address this question because of the low level of genomic redundancy of these enzymes, as opposed to mammals. Here, we systematically mutated the genes encoding heparan sulfate modification enzymes in C. elegans and analyzed their impact on excitatory and inhibitory neuromuscular junctions. Using single chain antibodies that recognize different heparan sulfate modification patterns, we show in vivo that these two heparan sulfate epitopes are carried by the SDN-1 core protein, the unique C. elegans syndecan orthologue, at neuromuscular junctions. Intriguingly, these antibodies differentially bind to excitatory and inhibitory synapses, implying unique heparan sulfate modification patterns at different neuromuscular junctions. Moreover, while most enzymes are individually dispensable for proper organization of neuromuscular junctions, we show that 3-O-sulfation of SDN-1 is required to maintain wild-type levels of the extracellular matrix protein MADD-4/Punctin, a central synaptic organizer that defines the identity of excitatory and inhibitory synaptic domains at the plasma membrane of muscle cells.

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