Ablation of sarcolipin results in atrial remodeling

Sarcolipin (SLN) is a key regulator of sarco(endo)plasmic reticulum (SR) Ca(2+)-ATPase (SERCA), and its expression is altered in diseased atrial myocardium. To determine the precise role of SLN in atrial Ca(2+) homeostasis, we developed a SLN knockout (sln-/-) mouse model and demonstrated that ablation of SLN enhances atrial SERCA pump activity. The present study is designed to determine the long-term effects of enhanced SERCA activity on atrial remodeling in the sln-/- mice. Calcium transient measurements show an increase in atrial SR Ca(2+) load and twitch Ca(2+) transients. Patch-clamping experiments demonstrate activation of the forward mode of sodium/calcium exchanger, increased L-type Ca(2+) channel activity, and prolongation of action potential duration at 90% repolarization in the atrial myocytes of sln-/- mice. Spontaneous Ca(2+) waves, delayed afterdepolarization, and triggered activities are frequent in the atrial myocytes of sln-/- mice. Furthermore, loss of SLN in atria is associated with increased interstitial fibrosis and altered expression of genes encoding collagen and other extracellular matrix proteins. Our results also show that the sln-/- mice are susceptible to atrial arrhythmias upon aging. Together, these findings indicate that ablation of SLN results in increased SERCA activity and SR Ca(2+) load, which, in turn, could cause abnormal intracellular Ca(2+) handling and atrial remodeling.     

Heat shock gene expression is regulated during teratocarcinoma cell differentiation and early embryonic development

Undifferentiated teratocarcinoma stem cells do not express heat shock genes. Solid teratocarcinomas grown in vivo which contain clusters of teratocarcinoma-derived differentiated tissue do respond to heat shock. During mouse embryonic development the expression of heat shock genes is first observed with morula/blastocyst stages of mouse primplantation embryos.     

Apactin is involved in remodeling of the actin cytoskeleton during regulated exocytosis

Apactin is an 80-kDa type I membrane glycoprotein derived from pro-Muclin, a precursor that also gives rise to the zymogen granule protein Muclin. Previous work showed that apactin is efficiently removed from the regulated secretory pathway and targeted to the actin-rich apical plasma membrane of the pancreatic acinar cell. The cytosolic tail (C-Tail) of apactin consists of 16 amino acids, has Thr casein kinase II and Ser protein kinase C phosphorylation sites, and a C-terminal PDZ-binding domain. Secretory stimulation of acinar cells causes a decrease in Thr phosphorylation and an increase in Ser phosphorylation of apactin. Fusion peptides of the C-Tail domain pulldown actin, ezrin, and EBP50/NHERF in a phosphorylation-dependent manner. HIV TAT-C-Tail fusion peptides were used as dominant negative constructs on living pancreatic cells to study effects on the actin cytoskeleton. During secretory stimulation, TAT-C-Tail-Thr/Asp phosphomimetic peptide caused an increase in actin-coated zymogen granules at the apical surface, while TAT-C-Tail-S/D phosphomimetic peptide caused a broadening of the actin cytoskeleton. These data indicate that stimulation-mediated Thr dephosphorylation allows decreased association of apactin with EBP50/NHERF and fosters actin remodeling to coat zymogen granules. Stimulation-mediated Ser phosphorylation increases apactin association with the actin cytoskeleton, maintaining tight bundling of actin microfilaments at the apical surface. Thus, apactin is involved in remodeling the apical cytoskeleton during regulated exocytosis in a manner controlled by phosphorylation of the apactin C-Tail.     

Mitochondrial genome content is regulated during nematode development

Growth and development rely on the mitochondrial respiratory chain (MRC) as the major source of ATP. We measured the mitochondrial DNA (mtDNA) copy number of each of the Caenorhabditis elegans developmental stages. Embryos, L1, L2, and L3 larvae all have approximately 25,000 copies of maternally derived mtDNA. The copy number increases fivefold in L4 larvae and a further sixfold in adult hermaphrodites, but only twofold in adult males. The majority of mtDNA in adult worms is germline associated, and germline-deficient mutants show markedly reduced mtDNA contents. With sperm-deficient or oocyte-deficient mutants, we confirm that mtDNA amplification is primarily associated with oocyte production. When mtDNA replication is inhibited, a quantitative and homogeneous arrest as L3 larvae occurs. Thus, mtDNA amplification is a necessary component of normal development and its regulation may involve an energy-sensing decision or checkpoint that can be invoked when mitochondrial energy generation is impaired.     

Stress resilience is established during development and is regulated by complement factors

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OLFML3 expression is decreased during prenatal muscle development and regulated by microRNA-155 in pigs

The Olfactomedin-like 3 (OLFML3) gene has matrix-related function involved in embryonic development. MicroRNA-155 (miR-155), 21- to 23-nucleotides (nt) noncoding RNA, regulated myogenesis by target mRNA. Our LongSAGE analysis suggested that OLFML3 gene was differently expressed during muscle development in pig. In this study, we cloned the porcine OLFML3 gene and detected its tissues distribution in adult Tongcheng pigs and dynamical expression in developmental skeletal muscle (12 prenatal and 10 postnatal stages) from Landrace (lean-type) and Tongcheng (obese-type) pigs. Subsequently, we analyzed the interaction between OLFML3 and miR-155. The OLFML3 was abundantly expressed in liver and pancreas, moderately in lung, small intestine and placenta, and weakly in other tissues and postnatal muscle. There were different dynamical expression patterns between Landrace and Tongcheng pigs during prenatal skeletal muscle development. The OLFML3 was down-regulated (33-50 days post coitus, dpc), subsequently up-regulated (50-70 dpc), and then down-regulated (70-100 dpc) in Landrace pigs, while in Tongcheng pigs, it was down-regulated (33-50 dpc), subsequently up-regulated (50-55 dpc) and then down-regulated (55-100 dpc). There was higher expression in Tongcheng than Landrace in prenatal muscle from 33 to 60 dpc, and opposite situation from 65 to 100 dpc. Dual luciferase assay and real time PCR documented that OLFML3 expression was regulated by miR-155 at mRNA level. Our research indicated that OLFML3 gene may affect prenatal skeletal muscle development and was regulated by miR-155. These finding will help understanding biological function and expression regulation of OLFML3 gene in mammal animals.     

CDKL5 expression is modulated during neuronal development and its subcellular distribution is tightly regulated by the C-terminal tail

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with Rett syndrome (RTT), West syndrome, and X-linked infantile spasms, sharing the common feature of mental retardation and early seizures. CDKL5 is a rather uncharacterized kinase, but its involvement in RTT seems to be explained by the fact that it works upstream of MeCP2, the main cause of Rett syndrome. To understand the role of this kinase for nervous system functions and to address if molecular mechanisms are involved in regulating its distribution and activity, we studied the ontogeny of CDKL5 expression in developing mouse brains by immunostaining and Western blotting. The expression profile of CDKL5 was compared with that of MeCP2. The two proteins share a general expression profile in the adult mouse brain, but CDKL5 levels appear to be highly modulated at the regional level. Its expression is strongly induced in early postnatal stages, and in the adult brain CDKL5 is present in mature neurons, but not in astroglia. Interestingly, the presence of CDKL5 in the cell nucleus varies at the regional level of the adult brain and is developmentally regulated. CDKL5 shuttles between the cytoplasm and the nucleus and the C-terminal tail is involved in localizing the protein to the cytoplasm in a mechanism depending on active nuclear export. Accordingly, Rett derivatives containing disease-causing truncations of the C terminus are constitutively nuclear, suggesting that they might act as gain of function mutations in this cellular compartment.     

VEGF expression is developmentally regulated during human brain angiogenesis

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor working as an endothelial cell-specific mitogen and exerting a trophic effect on neurons and glial cells, both these activities being essential during central nervous system vascularisation, development and repair. The vascularisation of human telencephalon takes place by means of an angiogenic mechanism, which starts at the beginning of corticogenesis and actively proceeds up to the last neuronal migration, when the basic scheme of the vascular network has been drawn. Our study focused on VEGF during this critical developmental period with the aim of identifying the cells that express VEGF and of correlating the events of angiogenesis with the main events of cerebral cortex formation. The results show that in fetal human brain VEGF protein is located on multiple cell types, cells proper to the nervous tissue, neuroepithelial cells, neuroblasts and radial glia cells, and non-neuronal cells, endothelial and periendothelial cells. In these cells VEGF expression appears developmentally regulated and is correlated with angiogenesis, which in turn responds to the high metabolic demands of the differentiating neocortex.     

TACC3 expression is tightly regulated during early differentiation

Transforming acidic coiled-coil (TACC) proteins are hypothesized to play a role in normal cellular growth and differentiation and to be involved in centrosomal microtubule stabilization. Our current studies aim to delineate the expression pattern of TACC3 protein during cellular differentiation and in a variety of normal human tissues. TACC3 is known to be upregulated in differentiating erythroid progenitor cells following treatment with erythropoietin and is required for replication of hematopoietic stem cells. However, we demonstrate that a dramatic upregulation of TACC3 also occurs during the early differentiation of NIH 3T3-L1 cells into adipocytes and PC12 cells into neurons, indicating that TACC3 mediates cellular differentiation in several cell types. Using real-time PCR, we quantitated the mRNA levels of TACC3 compared to TACC1 and TACC2 in various human adult tissues. We observed the highest expression of TACC3 mRNA in testis, spleen, thymus and peripheral blood leukocytes, all tissues undergoing high rates of differentiation, and a lower level of expression in ovary, prostate, pancreas, colon, small intestine, liver and kidney. In contrast, TACC1 and TACC2 mRNA levels are more widespread. By immunohistochemistry, we confirm that the TACC3 protein localizes to differentiating cell types, including spermatocytes, oocytes, epithelial cells, bone marrow cells and lymphocytes. Thus, these observations are concordant with a basic role for TACC3 during early stages of differentiation in normal tissues.     

Coronary development is regulated by ATP-dependent SWI/SNF chromatin remodeling component BAF180

Dissecting the molecular mechanisms that guide the proper development of epicardial cell lineages is critical for understanding the etiology of both congenital and adult forms of human cardiovascular disease. In this study, we describe the function of BAF180, a polybromo protein in ATP-dependent SWI/SNF chromatin remodeling complexes, in coronary development. Ablation of BAF180 leads to impaired epithelial-to-mesenchymal-transition (EMT) and arrested maturation of epicardium around E11.5. Three-dimensional collagen gel assays revealed that the BAF180 mutant epicardial cells indeed possess significantly compromised migrating and EMT potentials. Consequently, the mutant hearts form abnormal surface nodules and fail to develop the fine and continuous plexus of coronary vessels that cover the entire ventricle around E14. PECAM and α-SMA staining assays indicate that these nodules are defective structures resulting from the failure of endothelial and smooth muscle cells within them to form coronary vessels. PECAM staining also reveal that there are very few coronary vessels inside the myocardium of mutant hearts. Consistent with this, quantitative RT-PCR analysis indicate that the expression of genes involved in FGF, TGF, and VEGF pathways essential for coronary development are down-regulated in mutant hearts. Together, these data reveal for the first time that BAF180 is critical for coronary vessel formation.     

Alpha-actinin is required for tightly regulated remodeling of the actin cortical network during cytokinesis

Localization of the actin crosslinking protein, alpha-actinin, to the cleavage furrow has been previously reported. However, its functions during cytokinesis remain poorly understood. We have analyzed the functions of alpha-actinin during cytokinesis by a combination of molecular manipulations and imaging-based techniques. alpha-actinin gradually dissipated from the cleavage furrow as cytokinesis progressed. Overexpression of alpha-actinin caused increased accumulation of actin filaments because of inhibition of actin turnover, leading to cytokinesis failure. Global depletion of alpha-actinin by siRNA caused a decrease in the density of actin filaments throughout the cell cortex, surprisingly inducing accelerated cytokinesis and ectopic furrows. Local ablation of alpha-actinin induced accelerated cytokinesis specifically at the site of irradiation. Neither overexpression nor depletion of alpha-actinin had an apparent effect on myosin II organization. We conclude that cytokinesis in mammalian cells requires tightly regulated remodeling of the cortical actin network mediated by alpha-actinin in coordination with actomyosin-based cortical contractions.     

Dendrite-like process formation and cytoskeletal remodeling regulated by delta-catenin expression

Actin- and microtubule-mediated changes in cell shape are essential for many cellular activities. However, the molecular mechanisms underlying the interplay between the two are complex and remain obscure. Here we show that the expression of delta-catenin (or NPRAP/Neurojungin), a member of p120(ctn) subfamily of armadillo proteins can induce the branching of dendrite-like processes in 3T3 cells and enhance dendritic morphogenesis in primary hippocampal neurons. This induction of branching phenotype involves initially the disruption of filamentous actin, and requires the growth of microtubules. The carboxyl-terminal truncation mutant of delta-catenin can cluster and redistribute the full-length protein, and dominantly inhibit its branching effect. delta-Catenin forms protein complexes and can bind directly to actin in vitro. The carboxyl-terminal truncation of delta-catenin does not interfere with its actin-binding capability; therefore the actin interaction alone is not sufficient for the induction of dendrite-like processes. When delta-catenin-transformed cells establish elaborate dendrite-like branches, the main cellular processes become stabilized and resist the disruption of both actin filaments and microtubules, as determined by fluorescent light microscopy and time-lapse recording analyses. We suggest that delta-catenin can effect a biphasic cytoskeletal remodeling event which differentially regulates actin and microtubules and promotes cellular morphogenesis.