The microtubule modulator RanBP10 plays a critical role in regulation of platelet discoid shape and degranulation

Blood ◽  
2009 ◽  
Vol 114 (27) ◽  
pp. 5532-5540 ◽  
Author(s):  
Stefan Kunert ◽  
Imke Meyer ◽  
Silke Fleischhauer ◽  
Martin Wannack ◽  
Janine Fiedler ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Critical Role ◽  
Mutant Mice ◽  
Axis Ratio ◽  
Prolonged Bleeding Time ◽  
Normal Platelet ◽  
And Function ◽  
Granule Release

Abstract Terminally mature megakaryocytes undergo dramatic cellular reorganization to produce hundreds of virtually identical platelets. A hallmark feature of this process is the generation of an elaborate system of branched protrusions called proplatelets. We recently identified RanBP10 as a tubulin-binding protein that is concentrated along polymerized microtubules in mature megakaryocytes. RanBP10 depletion in vitro caused the disturbance of polymerized filaments. Here we study the function of RanBP10 in vivo by generating deficient mice using a gene-trap approach. Mutant mice show normal platelet counts, and fetal liver-derived megakaryocytes reveal only slightly reduced proplatelet formation. However, ultrastructural analysis unveiled a significantly increased geometric axis ratio for resting platelets, and many platelets exhibited disorders in microtubule filament numbers and localization. Mutant mice showed a markedly prolonged bleeding time. Granule release, a process that depends on internal contraction of the microtubule marginal coil, also was reduced. Flow cytometry analysis revealed reduced expression of CD62P and CD63 after PAR4-peptide stimulation. These data suggest that RanBP10 plays an essential role in hemostasis and in maintaining microtubule dynamics with respect to both platelet shape and function.

scholarly journals Reading and Function of a Histone Code Involved in Targeting Corepressor Complexes for Repression

2005 ◽  
Vol 25 (1) ◽  
pp. 324-335 ◽  
Author(s):  
Ho-Geun Yoon ◽  
Youngsok Choi ◽  
Philip A. Cole ◽  
Jiemin Wong
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Hormone Receptors ◽  
Critical Role ◽  
Pericentric Heterochromatin ◽  
Histone Code ◽  
Stable Association ◽  
And Function

ABSTRACT A central question in histone code theory is how various codes are recognized and utilized in vivo. Here we show that TBL1 and TBLR1, two WD-40 repeat proteins in the corepressor SMRT/N-CoR complexes, are functionally redundant and essential for transcriptional repression by unliganded thyroid hormone receptors (TR) but not essential for transcriptional activation by liganded TR. TBL1 and TBLR1 bind preferentially to hypoacetylated histones H2B and H4 in vitro and have a critical role in targeting the corepressor complexes to chromatin in vivo. We show that targeting SMRT/N-CoR complexes to the deiodinase 1 gene (D1) requires at least two interactions, one between unliganded TR and SMRT/N-CoR and the other between TBL1/TBLR1 and hypoacetylated histones. Neither interaction alone is sufficient for the stable association of the corepressor complexes with the D1 promoter. Our data support a feed-forward working model in which deacetylation exerted by initial unstable recruitment of SMRT/N-CoR complexes via their interaction with unliganded TR generates a histone code that serves to stabilize their own recruitment. Similarly, we find that targeting of the Sin3 complex to pericentric heterochromatin may also follow this model. Our studies provide an in vivo example that a histone code is not read independently but is recognized in the context of other interactions.

scholarly journals A critical role of VLA-4 in erythropoiesis in vivo

Blood ◽  
1996 ◽  
Vol 87 (6) ◽  
pp. 2513-2517 ◽  
Author(s):  
K Hamamura ◽  
H Matsuda ◽  
Y Takeuchi ◽  
S Habu ◽  
H Yagita ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Fetal Liver ◽  
Critical Role ◽  
In Utero ◽  
In Vitro Studies ◽  
Specific Interactions ◽  
Erythroid Progenitors

Hematopoiesis requires specific interactions with the microenvironments, and VLA-4 has been implicated in these interactions based on in vitro studies. To study the role of VLA-4 in hematopoiesis in vivo, we performed in utero treatment of mice with an anti-VLA-4 monoclonal antibody. Although all hematopoietic cells in fetal liver expressed VLA-4, the treatment specifically induced anemia. It had no effect on the development of nonerythroid lineage cells, including lymphoids and myeloids. In the treated liver almost no erythroblast was detected, whereas the erythroid progenitors, which give rise to erythroid colonies in vitro, were present. These results indicate that VLA-4 plays a critical role in erythropoiesis, while it is not critical in lymphopoiesis in vivo.

scholarly journals "Impotent" Platelets in Albinos With Prolonged Bleeding Times

Blood ◽  
1972 ◽  
Vol 39 (4) ◽  
pp. 490-499 ◽  
Author(s):  
Harold M. Maurer ◽  
James A. Wolff ◽  
Sue Buckingham ◽  
Arthur R. Spielvogel
Keyword(s):  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Bleeding Tendency ◽  
Prolonged Bleeding ◽  
Prolonged Bleeding Time ◽  
Normal Platelet ◽  
Tryptophan Metabolites ◽  
History Of

Abstract Functional, biochemical, and morphologic platelet abnormalities are reported in four children with the syndrome of albinism, mild bleeding tendency, prolonged bleeding time, and normal platelet count. In these children, primary platelet aggregation with adenosine diphosphate occurred normally, but secondary aggregation was impaired. Collagen and norepinephrine produced almost no platelet aggregation. Platelet content of serotonin (5-HT) was markedly reduced, and uptake and retention of 5-HT by the platelets in vivo and in vitro was poor. In one child who was given a tryptophan load, urinary tryptophan metabolites were normal, suggesting that there was no evidence of a block in the 5-HT synthetic pathway in the gastrointestinal tract. Electron microscopy revealed an absence of densely osmophilic granules in 5-HT poor platelets. Platelets from other albinos with no history of bleeding contained normal amounts of 5-HT and densely osmophilic granules.

scholarly journals Iκb Kinase α Is Essential for Mature B Cell Development and Function

2001 ◽  
Vol 193 (4) ◽  
pp. 417-426 ◽  
Author(s):  
Tsuneyasu Kaisho ◽  
Kiyoshi Takeda ◽  
Tohru Tsujimura ◽  
Taro Kawai ◽  
Fumiko Nomura ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Fetal Liver ◽  
Cell Development ◽  
B Cell Development ◽  
Cytokine Signaling ◽  
Iκb Kinase ◽  
And Function

IκB kinase (IKK) α and β phosphorylate IκB proteins and activate the transcription factor, nuclear factor (NF)-κB. Although both are highly homologous kinases, gene targeting experiments revealed their differential roles in vivo. IKKα is involved in skin and limb morphogenesis, whereas IKKβ is essential for cytokine signaling. To elucidate in vivo roles of IKKα in hematopoietic cells, we have generated bone marrow chimeras by transferring control and IKKα-deficient fetal liver cells. The mature B cell population was decreased in IKKα−/− chimeras. IKKα−/− chimeras also exhibited a decrease of serum immunoglobulin basal level and impaired antigen-specific immune responses. Histologically, they also manifested marked disruption of germinal center formation and splenic microarchitectures that depend on mature B cells. IKKα−/− B cells not only showed impairment of survival and mitogenic responses in vitro, accompanied by decreased, although inducible, NF-κB activity, but also increased turnover rate in vivo. In addition, transgene expression of bcl-2 could only partially rescue impaired B cell development in IKKα−/− chimeras. Taken together, these results demonstrate that IKKα is critically involved in the prevention of cell death and functional development of mature B cells.

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 Gut microbiota regulation of P-glycoprotein in the intestinal epithelium in maintenance of homeostasis

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Sage E. Foley ◽  
Christine Tuohy ◽  
Merran Dunford ◽  
Michael J. Grey ◽  
Heidi De Luca ◽  
...  
Keyword(s):  
Microbial Community ◽  
Intestinal Epithelium ◽  
Critical Role ◽  
Secondary Bile Acid ◽  
Core Microbiome ◽  
P Glycoprotein ◽  
Mucosal Barrier Function ◽  
And Function

Abstract Background P-glycoprotein (P-gp) plays a critical role in protection of the intestinal epithelia by mediating efflux of drugs/xenobiotics from the intestinal mucosa into the gut lumen. Recent studies bring to light that P-gp also confers a critical link in communication between intestinal mucosal barrier function and the innate immune system. Yet, despite knowledge for over 10 years that P-gp plays a central role in gastrointestinal homeostasis, the precise molecular mechanism that controls its functional expression and regulation remains unclear. Here, we assessed how the intestinal microbiome drives P-gp expression and function. Results We have identified a “functional core” microbiome of the intestinal gut community, specifically genera within the Clostridia and Bacilli classes, that is necessary and sufficient for P-gp induction in the intestinal epithelium in mouse models. Metagenomic analysis of this core microbial community revealed that short-chain fatty acid and secondary bile acid production positively associate with P-gp expression. We have further shown these two classes of microbiota-derived metabolites synergistically upregulate P-gp expression and function in vitro and in vivo. Moreover, in patients suffering from ulcerative colitis (UC), we find diminished P-gp expression coupled to the reduction of epithelial-derived anti-inflammatory endocannabinoids and luminal content (e.g., microbes or their metabolites) with a reduced capability to induce P-gp expression. Conclusion Overall, by means of both in vitro and in vivo studies as well as human subject sample analysis, we identify a mechanistic link between cooperative functional outputs of the complex microbial community and modulation of P-gp, an epithelial component, that functions to suppress overactive inflammation to maintain intestinal homeostasis. Hence, our data support a new cross-talk paradigm in microbiome regulation of mucosal inflammation.

scholarly journals Platelet storage at 22 degrees C: effect of type of agitation on morphology, viability, and function in vitro

Blood ◽  
1978 ◽  
Vol 52 (2) ◽  
pp. 425-435 ◽  
Author(s):  
S Holme ◽  
K Vaidja ◽  
S Murphy
Keyword(s):  
Platelet Count ◽  
Shape Change ◽  
Normal Platelet ◽  
Platelet Storage ◽  
Platelet Morphology ◽  
And Function ◽  
Change Response ◽  
Citrate Phosphate

Abstract Recovery in vivo after 51Cr labeling, platelet morphology, and platelet aggregation were studied with platelet concentrates (PC) stored for transfusion under carefully controlled conditions. PC were prepared to a final volume of 50 ml from whole blood anticoagulated with citrate- phosphate-dextrose (CPD). The platelet count was kept between 0.8 and 1.6 X 10(12) platelets/liter. The PC were stored in bags constructed of polyvinylchloride (PVC) or polyethylene (PE) at 22 degrees C for 72 hr. The bags were placed on a horizontal shaker or a ferris wheel for agitation during storage. No significant changes in pH or platelet count were observed during storage. PC stored on the wheel showed moderate loss of viability and a marked deterioration of platelet morphology and aggregation compared to the shaker. PC stored on the shaker in bags made of PE showed better aggregation with ADP and thrombin but had the same viability and morphology as PC in bags constructed of PVC. Maintenance of normal platelet morphology as determined by phase-contrast microscopy, extent of shape change response, and the size distribution according to the Coulter Counter correlated with recovery in vivo.

scholarly journals Platelet storage at 22 degrees C: effect of type of agitation on morphology, viability, and function in vitro

Blood ◽  
1978 ◽  
Vol 52 (2) ◽  
pp. 425-435 ◽  
Author(s):  
S Holme ◽  
K Vaidja ◽  
S Murphy
Keyword(s):  
Platelet Count ◽  
Shape Change ◽  
Normal Platelet ◽  
Platelet Storage ◽  
Platelet Morphology ◽  
And Function ◽  
Change Response ◽  
Citrate Phosphate

Recovery in vivo after 51Cr labeling, platelet morphology, and platelet aggregation were studied with platelet concentrates (PC) stored for transfusion under carefully controlled conditions. PC were prepared to a final volume of 50 ml from whole blood anticoagulated with citrate- phosphate-dextrose (CPD). The platelet count was kept between 0.8 and 1.6 X 10(12) platelets/liter. The PC were stored in bags constructed of polyvinylchloride (PVC) or polyethylene (PE) at 22 degrees C for 72 hr. The bags were placed on a horizontal shaker or a ferris wheel for agitation during storage. No significant changes in pH or platelet count were observed during storage. PC stored on the wheel showed moderate loss of viability and a marked deterioration of platelet morphology and aggregation compared to the shaker. PC stored on the shaker in bags made of PE showed better aggregation with ADP and thrombin but had the same viability and morphology as PC in bags constructed of PVC. Maintenance of normal platelet morphology as determined by phase-contrast microscopy, extent of shape change response, and the size distribution according to the Coulter Counter correlated with recovery in vivo.

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.

Maturation Stage-Specific Regulation of Megakaryocytic Genes by Pointed-Domain Ets Proteins.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1737-1737
Author(s):  
Liyan Pang ◽  
Xun Wang ◽  
Yuhuan Wang ◽  
Gerd Blobel ◽  
Mortimer Poncz
Keyword(s):  
Gene Expression ◽  
Fetal Liver ◽  
Critical Role ◽  
Regulatory Region ◽  
Maturation Stage ◽  
Specific Gene ◽  
Mrna Levels ◽  
Specific Regulation

Abstract The pointed-domain Ets transcription factor Fli-1 has a critical role during megakaryocyte-specific gene expression. Previously, we demonstrated that Fli-1 occupies the early megakaryocyte-specific gene αIIb in vivo. Moreover, our work suggested a mechanism for Fli-1 function by showing that Fli-1 facilitates GATA-1/FOG-1 dependent expression of the αIIb gene. However, studies by others with a targeted disruption of the Fli-1 gene in mice showed that while Fli-1 is essential for normal megakaryocyte maturation, αIIb mRNA levels were not significantly reduced in the resulting megakaryocytes, suggesting that a related Ets factor(s) might compensate for the loss of Fli-1. Here we show that the widely expressed pointed domain Ets protein GABPα specifically binds in vitro to Ets elements from two early megakaryocyte-specific genes, αIIb and c-mpl. Chromatin immunoprecipitation (ChIP) experiments using primary murine fetal liver-derived megakaryocytes reveal that GABPα associates with αIIb and c-mpl in vivo. Moreover, GABPα is capable of mediating GATA-1/FOG-1 synergy in the context of αIIb promoter constructs. These results suggest that GABPα contributes to megakaryocyte-restricted gene expression and is capable of at least partially compensating for the loss of Fli-1. However, loss of Fli-1 leads to a pronounced decrease in the expression of the late megakaryocyte-specific gene GPIX, indicating that compensation by GABPα is incomplete. Consistent with this observation, ChIP experiments fail to detect significant levels of GABPα at the regulatory region of GPIX while Fli-1 is readily detected there. Together, these results point to a model in which Fli-1 and GABPα serve overlapping, but distinct roles, during the development of megakaryocytes. GABPα may be important during early megakaryopoiesis, but Fli-1 exerting an essential role during late stages of maturation.

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