17 research outputs found
Microglia Control Vascular Architecture via a TGFβ1 Dependent Paracrine Mechanism Linked to Tissue Mechanics
© 2020, The Author(s). Tissue microarchitecture and mechanics are important in development and pathologies of the Central Nervous System (CNS); however, their coordinating mechanisms are unclear. Here, we report that during colonization of the retina, microglia contacts the deep layer of high stiffness, which coincides with microglial bipolarization, reduction in TGFβ1 signaling and termination of vascular growth. Likewise, stiff substrates induce microglial bipolarization and diminish TGFβ1 expression in hydrogels. Both microglial bipolarization in vivo and the responses to stiff substrates in vitro require intracellular adaptor Kindlin3 but not microglial integrins. Lack of Kindlin3 causes high microglial contractility, dysregulation of ERK signaling, excessive TGFβ1 expression and abnormally-patterned vasculature with severe malformations in the area of photoreceptors. Both excessive TGFβ1 signaling and vascular defects caused by Kindlin3-deficient microglia are rescued by either microglial depletion or microglial knockout of TGFβ1 in vivo. This mechanism underlies an interplay between microglia, vascular patterning and tissue mechanics within the CNS
Loss of R2D2 Proteins ROPN1 and ROPN1L Causes Defects in Murine Sperm Motility, Phosphorylation, and Fibrous Sheath Integrity1
Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility.
Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function
β3 phosphorylation of platelet αIIbβ3 is crucial for stability of arterial thrombus and microparticle formation in vivo
Abstract Background It is well accepted that functional activity of platelet integrin αIIbβ3 is crucial for hemostasis and thrombosis. The β3 subunit of the complex undergoes tyrosine phosphorylation shown to be critical for outside-in integrin signaling and platelet clot retraction ex vivo. However, the role of this important signaling event in other aspects of prothrombotic platelet function is unknown. Method Here, we assess the role of β3 tyrosine phosphorylation in platelet function regulation with a knock-in mouse strain, where two β3 cytoplasmic tyrosines are mutated to phenylalanine (DiYF). We employed platelet transfusion technique and intravital microscopy for observing the cellular events involved in specific steps of thrombus growth to investigate in detail the role of β3 tyrosine phosphorylation in arterial thrombosis in vivo. Results Upon injury, DiYF mice exhibited delayed arterial occlusion and unstable thrombus formation. The mean thrombus volume in DiYF mice formed on collagen was only 50% of that in WT. This effect was attributed to DiYF platelets but not to other blood cells and endothelium, which also carry these mutations. Transfusion of isolated DiYF but not WT platelets into irradiated WT mice resulted in reversal of the thrombotic phenotype and significantly prolonged blood vessel occlusion times. DiYF platelets exhibited reduced adhesion to collagen under in vitro shear conditions compared to WT platelets. Decreased platelet microparticle release after activation, both in vitro and in vivo, were observed in DiYF mice compared to WT mice. Conclusion β3 tyrosine phosphorylation of platelet αIIbβ3 regulates both platelet pro-thrombotic activity and the formation of a stable platelet thrombus, as well as arterial microparticle release
Targeted Disruption of Glycogen Synthase Kinase 3a (Gsk3a) in Mice Affects Sperm Motility Resulting in Male Infertility1
Microcystin pulldown of sperm PP2A.
<p><b>(A</b>) Caudal sperm extracts (2X10<sup>8</sup> sperm/ml) were incubated with microcystin- sepharose beads. The microcystin- bound proteins were eluted by boiling with Laemmli buffer and analyzed by Western blot with Anti-PP2A antibody (MC). 10μl of the input caudal sperm extract was loaded as a positive control (Input). Flow through (FT) obtained following the pull down, <i>i</i>.<i>e</i>. sperm extract left behind after incubation with microcystin sepharose beads, shows negligible levels of PP2A at the same control input loading volume. The same extracts were run as a duplicate blot and probed with Anti-PP1γ2 as a control for microcystin pull down. (<b>B)</b> Extracts from 5X10<sup>7</sup> sperm from each region of epididymis were subjected to microcystin pull down. Equal amounts of the microcystin bound proteins boiled in SDS- sample buffer were loaded in each lane and probed with anti-demethyl PP2A antibody. A duplicate blot probed with anti-PP2A antibody as control.</p
<i>In vivo</i> demethylation of sperm PP2A.
<p><b>(A)</b> Caudal sperm were incubated with 1 mM each of L-Homocysteine and Adenosine (L-Hcy + Ado), 1 mM L- Homocysteine (L-Hcy) or 1 mM adenosine (Ado) for 10 min. Sperm extracts were prepared by sonication and analyzed by western blot (2X10<sup>6</sup> sperm/lane) with anti-demethyl PP2A antibody and a duplicate blot probed with anti-PP2A antibody. Significant increase in levels of demethyl PP2A is observed on treatment with L-Homocysteine and adenosine (L-Hcy + Ado) compared to the untreated control sperm. (<b>B)</b> PP2A in extracts of L-Homocysteine and adenosine treated sperm was concentrated by microcystin pull down followed by western blot analysis of with anti-phosphotyrosine-PP2A (Y307) antibody shows increased levels of phosphorylated PP2A. (<b>C)</b> Caudal sperm incubated with 5 nM okadaic acid (OA), a concentration to specifically inhibit PP2A, also elevates demethyl PP2A and tyrosine phosphorylated PP2A as seen in <b>(D)</b> with 10<sup>7</sup> sperm/lane.</p
Demethylation of PP2A induced by alkali treatment.
<p>Microcystin beads incubated with sperm extracts from proximal caput, distal caput and caudal regions of epididymis were subjected to NaOH treatment (+). Equal amounts incubated without NaOH are untreated controls (-). Details of the procedure are described in Materials and Methods. Different volumes were loaded in Western blot for each epididymal region, hence shown as separate panels. Duplicate blots were processed, one was probed with anti-demethyl PP2A antibody and the other was probed with anti-PP2A antibody.</p
Catalytic activity of PP2A following its demethylation by L-homocysteine and adenosine treatment.
<p>Caudal sperm incubated with 1 mM L-homocysteine and adenosine were sonicated and the soluble protein fraction was collected. Protein phosphatase activity in this fraction was measured with phosphorylase <i>a</i> as the substrate. PP2A activity was measured as the activity that can be inhibited by 2nM OA. Demethylation of sperm PP2A by L-homocysteine and adenosine results in decreased total phosphatase and PP2A catalytic activity. The mean phosphatase activities from five sets of experiments are represented as mmol of PO4 released/minute/2x10<sup>5</sup> sperm ± SEM. ‘*’ denotes significant difference with P< 0.05. The demethylation in each experiment was confirmed by western blot analysis of the sperm extract.</p
LCMT1 and PME1 in sperm.
<p><b>A)</b> Western blot showing detectable levels of LCMT1 in mouse whole sperm extracts prepared in 1%SDS and testis extracts. Mouse brain extract was loaded as positive control for Anti-LCMT1 antibody. <b>B)</b> Presence of immunoreactive PME1 at ~43kd in HB+ sonicated soluble bull sperm extracts from all three regions of epididymis is shown with Anti-PME1 antibody. The blot was stripped and probed with Anti-PP2A antibody to show equal protein loading. <b>(C)</b> Sperm isolated from the three regions of the epididymis were incubated with DMSO (control) or 500 nM ABL127 for 1hr. Following this incubation, sperm extracts were prepared and analyzed by Western blot with anti-demethyl PP2A antibody. The first panel shows 10<sup>6</sup> proximal caput sperm/lane at 10 seconds exposure. The remaining panels show 2X10<sup>6</sup> distal caput or caudal sperm/lane at 60 seconds exposure. A duplicate blot probed with Anti-PP2A is used as loading control.</p