scholarly journals Knockdown of V-ATPase subunit A (atp6v1a) impairs acid secretion and ion balance in zebrafish (Danio rerio)

2007 ◽  
Vol 292 (5) ◽  
pp. R2068-R2076 ◽  
Author(s):  
Jiun-Lin Horng ◽  
Li-Yih Lin ◽  
Chang-Jen Huang ◽  
Fumi Katoh ◽  
Toyoji Kaneko ◽  
...  
Keyword(s):  
Acid Secretion ◽  
Apical Membrane ◽  
Critical Role ◽  
Ion Balance ◽  
Atpase Subunit ◽  
Subunit A ◽  
Acid Secreting ◽  
And Function

In the skin of zebrafish embryo, the vacuolar H+-ATPase (V-ATPase, H+ pump) distributed mainly in the apical membrane of H+-pump-rich cells, which pump internal acid out of the embryo and function similarly to acid-secreting intercalated cells in mammalian kidney. In addition to acid excretion, the electrogenic H+ efflux via the H+-ATPases in the gill apical membrane of freshwater fish was proposed to act as a driving force for Na+ entry through the apical Na+ channels. However, convincing molecular physiological evidence in vivo for this model is still lacking. In this study, we used morpholino-modified antisense oligonucleotides to knockdown the gene product of H+-ATPase subunit A ( atp6v1a) and examined the phenotype of the mutants. The H+-ATPase knockdown embryos revealed several abnormalities, including suppression of acid-secretion from skin, growth retardation, trunk deformation, and loss of internal Ca2+ and Na+. This finding reveals the critical role of H+-ATPase in embryonic acid -secretion and ion balance, as well.

scholarly journals A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

1995 ◽  
Vol 15 (10) ◽  
pp. 5214-5225 ◽  
Author(s):  
A D Catling ◽  
H J Schaeffer ◽  
C W Reuter ◽  
G R Reddy ◽  
M J Weber
Keyword(s):  
Protein Interactions ◽  
Critical Role ◽  
Phosphorylation Site ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Serum Stimulation ◽  
And Function

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

scholarly journals Thymosin β4 is essential for thrombus formation by controlling the G-actin/F-actin equilibrium in platelets

Haematologica ◽  
2021 ◽  
Author(s):  
Inga Scheller ◽  
Sarah Beck ◽  
Vanessa Göb ◽  
Carina Gross ◽  
Raluca A. I. Neagoe ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Critical Role ◽  
Actin Dynamics ◽  
Thymosin Β4 ◽  
Clot Retraction ◽  
And Function ◽  
External Signals

Coordinated rearrangements of the actin cytoskeleton are pivotal for platelet biogenesis from megakaryocytes (MKs) but also orchestrate key functions of peripheral platelets in hemostasis and thrombosis, such as granule release, the formation of filopodia and lamellipodia, or clot retraction. Along with profilin (Pfn) 1, thymosin β4 (encoded by Tmsb4x) is one of the two main G-actin sequestering proteins within cells of higher eukaryotes, and its intracellular concentration is particularly high in cells that rapidly respond to external signals by increased motility, such as platelets. Here, we analyzed constitutive Tmsb4x knockout (KO) mice to investigate the functional role of the protein in platelet production and function. Thymosin β4 deficiency resulted in a macrothrombocytopenia with only mildly increased platelet volume and an unaltered platelet life span. MK numbers in the bone marrow (BM) and spleen were unaltered, however, Tmsb4x KO MKs showed defective proplatelet formation in vitro and in vivo. Thymosin β4 deficient platelets displayed markedly decreased G-actin levels and concomitantly increased F-actin levels resulting in accelerated spreading on fibrinogen and clot retraction. Moreover, Tmsb4x KO platelets showed activation defects and an impaired immunoreceptor tyrosine-based activation motif (ITAM) signaling downstream of the activating collagen receptor glycoprotein (GP) VI. These defects translated into impaired aggregate formation under flow, protection from occlusive arterial thrombus formation in vivo and increased tail bleeding times. In summary, these findings point to a critical role of thymosin β4 for actin dynamics during platelet biogenesis, platelet activation downstream of GPVI and thrombus stability.

Flt3-Ligand Plays a Critical Role in Development and Function of CD8+/TCR− Facilitating Cells in Bone Marrow,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4003-4003
Author(s):  
Yiming Huang ◽  
Thomas Miller ◽  
Hong Xu ◽  
Yujie Wen ◽  
Suzanne T Ildstad
Keyword(s):  
Bone Marrow ◽  
Critical Role ◽  
Flt3 Ligand ◽  
Wild Type ◽  
Graft Versus Host ◽  
Equity Ownership ◽  
Total Body ◽  
And Function

Abstract Abstract 4003 Graft facilitating cells (FC) are a CD8+/TCR− bone marrow subpopulation that enhance engraftment of purified hematopoietic cells (HSC) in allogeneic mouse recipients without causing graft-versus-host disease. They also enhance engraftment of suboptimal numbers of syngeneic HSC. FC induce antigen-specific CD4+/CD25+/FoxP3+ regulatory T cells in vivo. The major subpopulation in FC is resembles plasmacytoid precursor dendritic cells (p-preDC) both phenotypically and functionally. Treatment of mice with Flt3 ligand (FL) results in a significant increase in FC in peripheral blood (PB) and FL-expanded-PB FC enhanced HSC engraftment. In this study, we evaluated the role of FL in FC development using FL-KO mice. We first compared FC from FL-KO B6 mice with FC from B6 mice to evaluate the FC total cellular composition. The number of FC was significantly decreased in FL-KO mice compared to wild type controls (P = 0.0003). The number of p-preDC FC was also significantly decreased (P = 0.0001), suggesting that FL is important in the development of p-preDC FC. Next, we tested whether FL-KO FC facilitate engraftment of HSC in allogeneic recipients. FC were sorted from FL-KO B6 mice and HSC (C-Kit+/Sca-1+/Lin−) were sorted from B6 mice. 10,000 B6 HSC plus 30,000 FL-KO FC were transplanted into NOD recipients conditioned with 950 cGy of total body irradiation. Controls received 10,000 B6 HSC with or without 30,000 B6 FC. Only 36% (5 of 14) NOD recipients of B6 HSC alone engrafted and two mice survived up to 160 days (Figure). Sixty-three percent (5 of 8) of recipients transplanted with B6 HSC + FL-KO B6 FC engrafted and only one mouse survived up to 160 days. Seventy-five percent (9 of 12) recipients of B6 HSC + B6 FC engrafted and seven of the mice survived more than 160 days. The level of donor chimerism in recipients of B6 HSC + B6 FC (57% ± 10%) was significantly higher than recipients of B6 HSC + FL-KO B6 FC (14% ± 3%; P = 0.003) or B6 HSC alone (22% ± 6%; P = 0.005). These data demonstrate that FL-KO FC fail to facilitate durable allogeneic HSC engraftment, suggesting that flt3-ligand plays a critical role in development of functional FC. Disclosures: Ildstad: Regenerex, LLC: Equity Ownership.

Developing Brain and Heart Cells Respond Similarly to Altered Mechanical Loads

2010 ◽  
Author(s):  
Benjamen A. Filas ◽  
Philip V. Bayly ◽  
Larry A. Taber
Keyword(s):  
Critical Role ◽  
Congenital Defects ◽  
Heart Cells ◽  
Morphological Adaptation ◽  
Mechanical Loads ◽  
Mechanical Environment ◽  
And Function ◽  
Internal Pressures

Past studies have shown that the mechanical environment plays a critical role in regulating tissue development and function. For example, in the embryonic heart abnormal internal pressures cause morphological adaptation leading to aberrant morphogenesis [1]. Similarly, increasing luminal pressure in the early brain results in hyper-proliferation of the neuroepithelium [2]. Less is known, however, about how embryonic precursor cells quantitatively adapt to changes in loading, especially in vivo and across tissue types. These data would be valuable in determining the role of altered mechanical loads in congenital defects.

Regulation of huntingtin palmitoylation and its role in Huntington Diseases

2007 ◽  
Vol 30 (4) ◽  
pp. 99
Author(s):  
F BJ Young ◽  
M R Hayden
Keyword(s):  
Health Research ◽  
Neurodegenerative Disorder ◽  
Critical Role ◽  
Neuronal Cell ◽  
Causative Mutation ◽  
General Role ◽  
Wide Range ◽  
And Function

Huntington’s Disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, cognitive, and psychiatric deficits and selective neuronal cell death. The causative mutation in HD is an expansion of the N-terminal polyglutamine tract in huntingtin (htt), which results in altered trafficking of mutant htt and enhanced toxicity to striatal neurons. Post-translational modification by the lipid palmitate has been shown to play a critical role in the trafficking and function of many proteins, including htt. It has been previously demonstrated that huntingtin-interacting protein 14 (HIP14) is a palmitoyl transferase that palmitoylates htt. Previous characterization of HIP14 demonstrated a reduced interaction with mutant htt resulting in reduced palmitoylation, suggesting that palmitoylation may play a role in the pathogenesis of HD. Most recently, we have identified cysteine 214 as a major site of htt palmitoylation in the N-terminus of htt, close to the site of polyglutamine expansion. It was demonstrated that mutation of this site, rendering htt palmitoylation-resistant, results in increased neuronal toxicity, enhanced inclusion formation, and in altered trafficking of htt. Remarkably, mutation of the palmitoylation site in wild type htt also resulted in enhanced toxicity similar to that seen in mutant htt. Together, these previous studies suggest a critical role of palmitoylation in htt trafficking and function. Based on this preliminary work, we are characterizing the enzymatic regulation of huntingtin palmitoylation. Exploring htt palmitoylation in a number of existing and new mouse models imparts key insights into how this process is regulated in vivo. We are also exploring the relationship between palmitoylation and other post-translational modifications of htt. These studies will lead to an understanding of the regulation of palmitoylation of huntingtin in vivo, as well as setting the precedent to understand the general role of palmitoylation in a wide range of other human diseases. Ultimately, this may lead to identification of new therapeutic targets and treatments for patients. F.B.J.Y. is supported by a Canadian Institutes of Health Research Walter and Jessie Boyd & Charles Scriver - Child and Family Research Institute - UBC MD/PhD Studentship Award. She also receives funding from the Michael Smith Foundation for Health Research as a Junior Trainee.

scholarly journals The role of Mfn2 in the structure and function of ER-mitochondrial tethering in vivo

2021 ◽  
Author(s):  
Song Han ◽  
Fanpeng Zhao ◽  
Jeffrey Hsia ◽  
Xiaopin Ma ◽  
Yi Liu ◽  
...  
Keyword(s):  
Pyramidal Neurons ◽  
Critical Role ◽  
Biochemical Changes ◽  
Conditional Knockout ◽  
Functional Studies ◽  
And Function ◽  
Close Contacts

The mitochondria-ER contacts (MERCs) plays an essential role in multiple cell physiological process. While Mfn2 was the first protein implicated in the formation of MERCs, it is debated whether it acts as a tether or antagonizer, largely based on in vitro studies. To understand the role of Mfn2 in MERCs in vivo, we characterized ultrastructural and biochemical changes of MERCs in pyramidal neurons of hippocampus in Mfn2 conditional knockout (KO) mice and in Mfn2 overexpression (OE) mice and found Mfn2 ablation caused reduced close contacts while Mfn2 OE caused increased close contacts between ER and mitochondria in vivo. Functional studies on SH-SY5Y cells with Mfn2 KO or overexpression demonstrating similar biochemical changes found that mitochondrial calcium uptake along with IP3R3-Grp75 interaction was decreased in Mfn2 KO cells but increased in the Mfn2 OE cells. Lastly, we found Mfn2 KO decreased and Mfn2 OE increased the interaction between the ER-mitochondria tethering pair of VAPB-PTPIP51. In conclusion, our study supports the notion that Mfn2 plays a critical role in ER-mitochondrial tethering and the formation of close contacts in neuronal cells in vivo.

scholarly journals The role of the PZP domain of AF10 in acute leukemia driven by AF10 translocations

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Brianna J. Klein ◽  
Anagha Deshpande ◽  
Khan L. Cox ◽  
Fan Xuan ◽  
Mohamad Zandian ◽  
...  
Keyword(s):  
Critical Role ◽  
Cellular Transformation ◽  
Acute Leukemias ◽  
Hematopoietic Stem ◽  
Nucleosome Core ◽  
Stem And Progenitor Cells ◽  
Transforming Activity

AbstractChromosomal translocations of the AF10 (or MLLT10) gene are frequently found in acute leukemias. Here, we show that the PZP domain of AF10 (AF10PZP), which is consistently impaired or deleted in leukemogenic AF10 translocations, plays a critical role in blocking malignant transformation. Incorporation of functional AF10PZP into the leukemogenic CALM-AF10 fusion prevents the transforming activity of the fusion in bone marrow-derived hematopoietic stem and progenitor cells in vitro and in vivo and abrogates CALM-AF10-mediated leukemogenesis in vivo. Crystallographic, biochemical and mutagenesis studies reveal that AF10PZP binds to the nucleosome core particle through multivalent contacts with the histone H3 tail and DNA and associates with chromatin in cells, colocalizing with active methylation marks and discriminating against the repressive H3K27me3 mark. AF10PZP promotes nuclear localization of CALM-AF10 and is required for association with chromatin. Our data indicate that the disruption of AF10PZP function in the CALM-AF10 fusion directly leads to transformation, whereas the inclusion of AF10PZP downregulates Hoxa genes and reverses cellular transformation. Our findings highlight the molecular mechanism by which AF10 targets chromatin and suggest a model for the AF10PZP-dependent CALM-AF10-mediated leukemogenesis.

scholarly journals Essential role of IPS-1 in innate immune responses against RNA viruses

2006 ◽  
Vol 203 (7) ◽  
pp. 1795-1803 ◽  
Author(s):  
Himanshu Kumar ◽  
Taro Kawai ◽  
Hiroki Kato ◽  
Shintaro Sato ◽  
Ken Takahashi ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Rna Virus ◽  
Critical Role ◽  
Nuclear Factor Κb ◽  
Type I ◽  
Innate Immune Responses ◽  
Antiviral Responses ◽  
Deficient Mice

IFN-β promoter stimulator (IPS)-1 was recently identified as an adapter for retinoic acid–inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (Mda5), which recognize distinct RNA viruses. Here we show the critical role of IPS-1 in antiviral responses in vivo. IPS-1–deficient mice showed severe defects in both RIG-I– and Mda5-mediated induction of type I interferon and inflammatory cytokines and were susceptible to RNA virus infection. RNA virus–induced interferon regulatory factor-3 and nuclear factor κB activation was also impaired in IPS-1–deficient cells. IPS-1, however, was not essential for the responses to either DNA virus or double-stranded B-DNA. Thus, IPS-1 is the sole adapter in both RIG-I and Mda5 signaling that mediates effective responses against a variety of RNA viruses.

scholarly journals Brain-Specific SNAP-25 Deletion Leads to Elevated Extracellular Glutamate Level and Schizophrenia-Like Behavior in Mice

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Hua Yang ◽  
Mengjie Zhang ◽  
Jiahao Shi ◽  
Yunhe Zhou ◽  
Zhipeng Wan ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Mouse Model ◽  
Glutamate Release ◽  
Critical Role ◽  
Conditional Knockout ◽  
Snare Complex ◽  
Extracellular Glutamate ◽  
Glutamate Level

Several studies have associated reduced expression of synaptosomal-associated protein of 25 kDa (SNAP-25) with schizophrenia, yet little is known about its role in the illness. In this paper, a forebrain glutamatergic neuron-specific SNAP-25 knockout mouse model was constructed and studied to explore the possible pathogenetic role of SNAP-25 in schizophrenia. We showed that SNAP-25 conditional knockout (cKO) mice exhibited typical schizophrenia-like phenotype. A significantly elevated extracellular glutamate level was detected in the cerebral cortex of the mouse model. Compared with Ctrls, SNAP-25 was dramatically reduced by about 60% both in cytoplasm and in membrane fractions of cerebral cortex of cKOs, while the other two core members of SNARE complex: Syntaxin-1 (increased ~80%) and Vamp2 (increased ~96%) were significantly increased in cell membrane part. Riluzole, a glutamate release inhibitor, significantly attenuated the locomotor hyperactivity deficits in cKO mice. Our findings provide in vivo functional evidence showing a critical role of SNAP-25 dysfunction on synaptic transmission, which contributes to the developmental of schizophrenia. It is suggested that a SNAP-25 cKO mouse, a valuable model for schizophrenia, could address questions regarding presynaptic alterations that contribute to the etiopathophysiology of SZ and help to consummate the pre- and postsynaptic glutamatergic pathogenesis of the illness.

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