Publications by authors named "John H Hartwig"

83 Publications

Sarcomeric and nonmuscle α-actinin isoforms exhibit differential dynamics at skeletal muscle Z-lines.

Cytoskeleton (Hoboken) 2018 05 1;75(5):213-228. Epub 2018 Apr 1.

Division of Genetics and Genomics, The Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, 02115.

The α-actinin proteins are a highly conserved family of actin crosslinkers that mediate interactions between several cytoskeletal and sarcomeric proteins. Nonsarcomeric α-actinin-1 and α-actinin-4 crosslink actin filaments in the cytoskeleton, while sarcomeric α-actinin-2 and α-actinin-3 serve a crucial role in anchoring actin filaments to the muscle Z-line. To assess the difference in turnover dynamics and structure/function properties between the α-actinin isoforms at the sarcomeric Z-line, we used Fluorescence Recovery After Photobleaching (FRAP) in primary myofiber cultures. We found that the recovery kinetics of these proteins followed three distinct patterns: α-actinin-2/α-actinin-3 had the slowest turn over, α-actinin-1 recovered to an intermediate degree, and α-actinin-4 had the fastest recovery. Interestingly, the isoforms' patterns of recovery were reversed at adhesion plaques in fibroblasts. This disparity suggests that the different α-actinin isoforms have unique association kinetics in myofibers and that nonmuscle isoform interactions are more dynamic at the sarcomeric Z-line. Protein domain-specific investigations using α-actinin-2/4 chimeric proteins showed that differential dynamics between sarcomeric and nonmuscle isoforms are regulated by cooperative interactions between the N-terminal actin-binding domain, the spectrin-like linker region and the C-terminal calmodulin-like EF hand domain. Together, these findings demonstrate that α-actinin isoforms are unique in binding dynamics at the Z-line and suggest differentially evolved interactive and Z-line association capabilities of each functional domain.
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http://dx.doi.org/10.1002/cm.21442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943145PMC
May 2018

Synthesis and dephosphorylation of MARCKS in the late stages of megakaryocyte maturation drive proplatelet formation.

Blood 2016 Mar 7;127(11):1468-80. Epub 2016 Jan 7.

Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Boston, MA; Harvard Medical School, Boston, MA; Vascular Biology Program, Department of Surgery, Children's Hospital Boston, Boston, MA.

Platelets are essential for hemostasis, and thrombocytopenia is a major clinical problem. Megakaryocytes (MKs) generate platelets by extending long processes, proplatelets, into sinusoidal blood vessels. However, very little is known about what regulates proplatelet formation. To uncover which proteins were dynamically changing during this process, we compared the proteome and transcriptome of round vs proplatelet-producing MKs by 2D difference gel electrophoresis (DIGE) and polysome profiling, respectively. Our data revealed a significant increase in a poorly-characterized MK protein, myristoylated alanine-rich C-kinase substrate (MARCKS), which was upregulated 3.4- and 5.7-fold in proplatelet-producing MKs in 2D DIGE and polysome profiling analyses, respectively. MARCKS is a protein kinase C (PKC) substrate that binds PIP2. In MKs, it localized to both the plasma and demarcation membranes. MARCKS inhibition by peptide significantly decreased proplatelet formation 53%. To examine the role of MARCKS in the PKC pathway, we treated MKs with polymethacrylate (PMA), which markedly increased MARCKS phosphorylation while significantly inhibiting proplatelet formation 84%, suggesting that MARCKS phosphorylation reduces proplatelet formation. We hypothesized that MARCKS phosphorylation promotes Arp2/3 phosphorylation, which subsequently downregulates proplatelet formation; both MARCKS and Arp2 were dephosphorylated in MKs making proplatelets, and Arp2 inhibition enhanced proplatelet formation. Finally, we used MARCKS knockout (KO) mice to probe the direct role of MARCKS in proplatelet formation; MARCKS KO MKs displayed significantly decreased proplatelet levels. MARCKS expression and signaling in primary MKs is a novel finding. We propose that MARCKS acts as a "molecular switch," binding to and regulating PIP2 signaling to regulate processes like proplatelet extension (microtubule-driven) vs proplatelet branching (Arp2/3 and actin polymerization-driven).
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http://dx.doi.org/10.1182/blood-2015-08-663146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4797023PMC
March 2016

An adventitious interaction of filamin A with RhoGDI2(Tyr153Glu).

Biochem Biophys Res Commun 2016 Jan 17;469(3):659-64. Epub 2015 Dec 17.

Hematology Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston MA, USA. Electronic address:

Filamin A (FLNA) is an actin filament crosslinking protein with multiple intracellular binding partners. Mechanical force exposes cryptic FLNA binding sites for some of these ligands. To identify new force-dependent binding interactions, we used a fusion construct composed of two FLNA domains, one of which was previously identified as containing a force-dependent binding site as a bait in a yeast two-hybrid system and identified the Rho dissociation inhibitor 2 (RhoGDI2) as a potential interacting partner. A RhoGDI2 truncate with 81 N-terminal amino acid residues and a phosphomimetic mutant, RhoGDI(Tyr153Glu) interacted with the FLNA construct. However, neither wild-type or full-length RhoGDI2 phosphorylated at Y153 interacted with FLNA. Our interpretation of these contradictions is that truncation and/or mutation of RhoGDI2 perturbs its conformation to expose a site that adventitiously binds FLNA and is not a bona-fide interaction. Therefore, previous studies reporting that a RhoGDI(Y153E) mutant suppresses the metastasis of human bladder cancer cells must be reinvestigated in light of artificial interaction of this point mutant with FLNA.
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http://dx.doi.org/10.1016/j.bbrc.2015.12.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4715983PMC
January 2016

Platelet actin nodules are podosome-like structures dependent on Wiskott-Aldrich syndrome protein and ARP2/3 complex.

Nat Commun 2015 Jun 1;6:7254. Epub 2015 Jun 1.

Centre for Cardiovascular Sciences, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

The actin nodule is a novel F-actin structure present in platelets during early spreading. However, only limited detail is known regarding nodule organization and function. Here we use electron microscopy, SIM and dSTORM super-resolution, and live-cell TIRF microscopy to characterize the structural organization and signalling pathways associated with nodule formation. Nodules are composed of up to four actin-rich structures linked together by actin bundles. They are enriched in the adhesion-related proteins talin and vinculin, have a central core of tyrosine phosphorylated proteins and are depleted of integrins at the plasma membrane. Nodule formation is dependent on Wiskott-Aldrich syndrome protein (WASp) and the ARP2/3 complex. WASp(-/-) mouse blood displays impaired platelet aggregate formation at arteriolar shear rates. We propose actin nodules are platelet podosome-related structures required for platelet-platelet interaction and their absence contributes to the bleeding diathesis of Wiskott-Aldrich syndrome.
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http://dx.doi.org/10.1038/ncomms8254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4458878PMC
June 2015

FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets.

Blood 2015 Jul 2;126(1):80-8. Epub 2015 Apr 2.

Division of Hematology, Brigham and Women's Hospital, Boston, MA; Department of Medicine, Harvard Medical School, Boston, MA;

Bin-Amphiphysin-Rvs (BAR) and Fes-CIP4 homology BAR (F-BAR) proteins generate tubular membrane invaginations reminiscent of the megakaryocyte (MK) demarcation membrane system (DMS), which provides membranes necessary for future platelets. The F-BAR protein PACSIN2 is one of the most abundant BAR/F-BAR proteins in platelets and the only one reported to interact with the cytoskeletal and scaffold protein filamin A (FlnA), an essential regulator of platelet formation and function. The FlnA-PACSIN2 interaction was therefore investigated in MKs and platelets. PACSIN2 associated with FlnA in human platelets. The interaction required FlnA immunoglobulin-like repeat 20 and the tip of PACSIN2 F-BAR domain and enhanced PACSIN2 F-BAR domain membrane tubulation in vitro. Most human and wild-type mouse platelets had 1 to 2 distinct PACSIN2 foci associated with cell membrane GPIbα, whereas Flna-null platelets had 0 to 4 or more foci. Endogenous PACSIN2 and transfected enhanced green fluorescent protein-PACSIN2 were concentrated in midstage wild-type mouse MKs in a well-defined invagination of the plasma membrane reminiscent of the initiating DMS and dispersed in the absence of FlnA binding. The DMS appeared less well defined, and platelet territories were not readily visualized in Flna-null MKs. We conclude that the FlnA-PACSIN2 interaction regulates membrane tubulation in MKs and platelets and likely contributes to DMS formation.
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http://dx.doi.org/10.1182/blood-2014-07-587600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4492198PMC
July 2015

The Ashwell-Morell receptor regulates hepatic thrombopoietin production via JAK2-STAT3 signaling.

Nat Med 2015 Jan 8;21(1):47-54. Epub 2014 Dec 8.

Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

The hepatic Ashwell-Morell receptor (AMR) can bind and remove desialylated platelets. Here we demonstrate that platelets become desialylated as they circulate and age in blood. Binding of desialylated platelets to the AMR induces hepatic expression of thrombopoietin (TPO) mRNA and protein, thereby regulating platelet production. Endocytic AMR controls TPO expression through Janus kinase 2 (JAK2) and the acute phase response signal transducer and activator of transcription 3 (STAT3) in vivo and in vitro. Recognition of this newly identified physiological feedback mechanism illuminates the pathophysiology of platelet diseases, such as essential thrombocythemia and immune thrombocytopenia, and contributes to an understanding of the mechanisms of thrombocytopenia observed with JAK1/2 inhibition.
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http://dx.doi.org/10.1038/nm.3770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303234PMC
January 2015

Microtubule sliding drives proplatelet elongation and is dependent on cytoplasmic dynein.

Blood 2015 Jan 19;125(5):860-8. Epub 2014 Nov 19.

Division of Translational Medicine and.

Bone marrow megakaryocytes produce platelets by extending long cytoplasmic protrusions, designated proplatelets, into sinusoidal blood vessels. Although microtubules are known to regulate platelet production, the underlying mechanism of proplatelet elongation has yet to be resolved. Here we report that proplatelet formation is a process that can be divided into repetitive phases (extension, pause, and retraction), as revealed by differential interference contrast and fluorescence loss after photoconversion time-lapse microscopy. Furthermore, we show that microtubule sliding drives proplatelet elongation and is dependent on cytoplasmic dynein under static and physiological shear stress by using fluorescence recovery after photobleaching in proplatelets with fluorescence-tagged β1-tubulin. A refined understanding of the specific mechanisms regulating platelet production will yield strategies to treat patients with thrombocythemia or thrombocytopenia.
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http://dx.doi.org/10.1182/blood-2014-09-600858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4311231PMC
January 2015

Megakaryocyte-specific Profilin1-deficiency alters microtubule stability and causes a Wiskott-Aldrich syndrome-like platelet defect.

Nat Commun 2014 Sep 4;5:4746. Epub 2014 Sep 4.

1] Department of Experimental Biomedicine, University Hospital, University of Würzburg, Würzburg 97080, Germany [2] Rudolf Virchow Centre, University of Würzburg, Würzburg 97080, Germany.

Wiskott-Aldrich syndrome (WAS) is caused by mutations in the WAS gene and is characterized by immunodeficiency, eczema and microthrombocytopenia. The molecular link between WAS mutations and microthrombocytopenia is unknown. Profilin1 (Pfn1) is a key actin-regulating protein that, besides actin, interacts with phosphoinositides and multiple proline-rich proteins, including the WAS protein (WASp)/WASp-interacting protein (WIP) complex. Here we report that mice with a megakaryocyte/platelet-specific Pfn1 deficiency display microthrombocytopenia due to accelerated turnover of platelets and premature platelet release into the bone marrow. Both Pfn1-null mouse platelets and platelets isolated from WAS patients contained abnormally organized and hyperstable microtubules. These results reveal an unexpected function of Pfn1 as a regulator of microtubule organization and point to a previously unrecognized mechanism underlying the platelet formation defect in WAS patients.
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http://dx.doi.org/10.1038/ncomms5746DOI Listing
September 2014

Documentation and localization of force-mediated filamin A domain perturbations in moving cells.

Nat Commun 2014 Aug 14;5:4656. Epub 2014 Aug 14.

Translational Medicine Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02445, USA.

Endogenously and externally generated mechanical forces influence diverse cellular activities, a phenomenon defined as mechanotransduction. Deformation of protein domains by application of stress, previously documented to alter macromolecular interactions in vitro, could mediate these effects. We engineered a photon-emitting system responsive to unfolding of two repeat domains of the actin filament (F-actin) crosslinker protein filamin A (FLNA) that binds multiple partners involved in cell signalling reactions and validated the system using F-actin networks subjected to myosin-based contraction. Expressed in cultured cells, the sensor-containing FLNA construct reproducibly reported FLNA domain unfolding strikingly localized to dynamic, actively protruding, leading cell edges. The unfolding signal depends upon coherence of F-actin-FLNA networks and is enhanced by stimulating cell contractility. The results establish protein domain distortion as a bona fide mechanism for mechanotransduction in vivo.
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http://dx.doi.org/10.1038/ncomms5656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139033PMC
August 2014

Regulation of dynamin oligomerization in cells: the role of dynamin-actin interactions and its GTPase activity.

Traffic 2014 Aug 24;15(8):819-38. Epub 2014 Jun 24.

Division of Nephrology, Massachusetts General Hospital, Charlestown, MA, 02129, USA.

Dynamin is a 96-kDa protein that has multiple oligomerization states that influence its GTPase activity. A number of different dynamin effectors, including lipids, actin filaments, and SH3-domain-containing proteins, have been implicated in the regulation of dynamin oligomerization, though their roles in influencing dynamin oligomerization have been studied predominantly in vitro using recombinant proteins. Here, we identify higher order dynamin oligomers such as rings and helices in vitro and in live cells using fluorescence lifetime imaging microscopy (FLIM). FLIM detected GTP- and actin-dependent dynamin oligomerization at distinct cellular sites, including the cell membrane and transition zones where cortical actin transitions into stress fibers. Our study identifies a major role for direct dynamin-actin interactions and dynamin's GTPase activity in the regulation of dynamin oligomerization in cells.
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http://dx.doi.org/10.1111/tra.12178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4107071PMC
August 2014

Wiskott-Aldrich syndrome protein (WASp) controls the delivery of platelet transforming growth factor-β1.

J Biol Chem 2013 Nov 16;288(48):34352-63. Epub 2013 Oct 16.

From the Division of Translational Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115.

Platelets are immunologically competent cells containing cytokines such as TGF-β1 that regulate cell-mediated immunity. However, the mechanisms underlying cytokine secretion from platelets are undefined. The Wiskott-Aldrich syndrome protein (WASp) regulates actin polymerization in nucleated hematopoietic cells but has other role(s) in platelets. WASp-null (WASp(-/-)) platelets stimulated with a PAR-4 receptor agonist had increased TGF-β1 release compared with WT platelets; inhibiting WASp function with wiskostatin augmented TRAP-induced TGF-β1 release in human platelets. TGF-β1 release is dissociated from α-granule secretion (P-selectin up-regulation) and occurs more gradually, with ∼10-15% released after 30-60 min. Blockade of Src family kinase-mediated WASp Tyr-291/Tyr-293 phosphorylation increased TGF-β1 release, with no additive effect in WASp(-/-) platelets, signifying that phosphorylation is critical for WASp-limited TGF-β1 secretion. Inhibiting F-actin assembly with cytochalasin D enhanced secretion in WT platelets and further increased TGF-β1 release in WASp(-/-) platelets, indicating that WASp and actin assembly independently regulate TGF-β1 release. A permeabilized platelet model was used to test the role of upstream small GTPases in TGF-β1 release. N17Cdc42, but not Rac1 mutants, increased TGF-β1 secretion and abrogated WASp phosphorylation. We conclude that WASp function restricts TGF-β1 secretion in a Cdc42- and Src family kinase-dependent manner and independently of actin assembly.
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http://dx.doi.org/10.1074/jbc.M113.459750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3843050PMC
November 2013

Platelets lacking PIP5KIγ have normal integrin activation but impaired cytoskeletal-membrane integrity and adhesion.

Blood 2013 Apr 31;121(14):2743-52. Epub 2013 Jan 31.

Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.

Three isoforms of phosphatidylinositol-4-phosphate 5-kinase (PIP5KIα, PIP5KIβ, and PIP5KIγ) can each catalyze the final step in the synthesis of phosphatidylinositol-4,5-bisphosphate (PIP2), which in turn can be either converted to second messengers or bind directly to and thereby regulate proteins such as talin. A widely quoted model speculates that only p90, a longer splice form of platelet-specific PIP5KIγ, but not the shorter p87 PIP5KIγ, regulates the ligand-binding activity of integrins via talin. However, when we used mice genetically engineered to lack only p90 PIP5KIγ, we found that p90 PIP5KIγ is not critical for integrin activation or platelet adhesion on collagen. However, p90 PIP5KIγ-null platelets do have impaired anchoring of their integrins to the underlying cytoskeleton. Platelets lacking both the p90 and p87 PIP5KIγ isoforms had normal integrin activation and actin dynamics, but impaired anchoring of their integrins to the cytoskeleton. Most importantly, they formed weak shear-resistant adhesions ex vivo and unstable vascular occlusions in vivo. Together, our studies demonstrate that, although PIP5KIγ is essential for normal platelet function, individual isoforms of PIP5KIγ fulfill unique roles for the integrin-dependent integrity of the membrane cytoskeleton and for the stabilization of platelet adhesion.
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http://dx.doi.org/10.1182/blood-2012-07-445205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3617636PMC
April 2013

Differential remodeling of actin cytoskeleton architecture by profilin isoforms leads to distinct effects on cell migration and invasion.

Cancer Cell 2012 Nov;22(5):615-30

Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

Dynamic actin cytoskeletal reorganization is integral to cell motility. Profilins are well-characterized regulators of actin polymerization; however, functional differences among coexpressed profilin isoforms are not well defined. Here, we demonstrate that profilin-1 and profilin-2 differentially regulate membrane protrusion, motility, and invasion; these processes are promoted by profilin-1 and suppressed by profilin-2. Compared to profilin-1, profilin-2 preferentially drives actin polymerization by the Ena/VASP protein, EVL. Profilin-2 and EVL suppress protrusive activity and cell motility by an actomyosin contractility-dependent mechanism. Importantly, EVL or profilin-2 downregulation enhances invasion in vitro and in vivo. In human breast cancer, lower EVL expression correlates with high invasiveness and poor patient outcome. We propose that profilin-2/EVL-mediated actin polymerization enhances actin bundling and suppresses breast cancer cell invasion.
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http://dx.doi.org/10.1016/j.ccr.2012.09.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3500527PMC
November 2012

Myosin-X facilitates Shigella-induced membrane protrusions and cell-to-cell spread.

Cell Microbiol 2013 Mar 13;15(3):353-367. Epub 2012 Nov 13.

Department of Medicine, Division of Infectious Diseases, University of Florida College of Medicine, Gainesville, FL, USA.

The intracellular pathogen Shigella flexneri forms membrane protrusions to spread from cell to cell. As protrusions form, myosin-X (Myo10) localizes to Shigella. Electron micrographs of immunogold-labelled Shigella-infected HeLa cells reveal that Myo10 concentrates at the bases and along the sides of bacteria within membrane protrusions. Time-lapse video microscopy shows that a full-length Myo10 GFP-construct cycles along the sides of Shigella within the membrane protrusions as these structures progressively lengthen. RNAi knock-down of Myo10 is associated with shorter protrusions with thicker stalks, and causes a >80% decrease in confluent cell plaque formation. Myo10 also concentrates in membrane protrusions formed by another intracellular bacteria, Listeria, and knock-down of Myo10 also impairs Listeria plaque formation. In Cos7 cells (contain low concentrations of Myo10), the expression of full-length Myo10 nearly doubles Shigella-induced protrusion length, and lengthening requires the head domain, as well as the tail-PH domain, but not the FERM domain. The GFP-Myo10-HMM domain localizes to the sides of Shigella within membrane protrusions and the GFP-Myo10-PH domain localizes to host cell membranes. We conclude thatMyo10 generates the force to enhance bacterial-induced protrusions by binding its head region to actin filaments and its PH tail domain to the peripheral membrane.
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http://dx.doi.org/10.1111/cmi.12051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4070382PMC
March 2013

Electron microscopy and 3D reconstruction reveals filamin Ig domain binding to F-actin.

J Mol Biol 2012 Dec 4;424(5):248-56. Epub 2012 Oct 4.

Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA 02118, USA.

Filamin A (FLNa) is an actin-binding protein that cross-links F-actin into networks of orthogonally branched filaments. FLNa also directs the networks to integrins while responding to mechanochemical signaling pathways. Flexible, 160-nm-long FLNa molecules are tail-to-tail dimers, each subunit of which contains an N-terminal calponin homology (CH)/actin-binding domain connected by a series of 24 immunoglobulin (Ig) repeats to a dimerization site at their C-terminal end. Whereas the contribution of the CH domains to F-actin affinity is weak (apparent K(a)~10(5)), the binding of the intact protein to F-actin is strong (apparent K(a)~10(8)), suggesting involvement of additional parts of the molecule in this association. Indeed, previous results indicate that Ig repeats along FLNa contribute significantly to the strength of the actin filament interaction. In the current study, we used electron microscopy and three-dimensional reconstruction to elucidate the structural basis of the Ig repeat-F-actin binding. We find that FLNa density is clearly delineated in reconstructions of F-actin complexed either with a four-Ig-repeat segment of FLNa containing Ig repeat 10 or with immunoglobulin-like filamin A repeat (IgFLNa)10 alone. The mass attributable to IgFLNa10 lies peripherally along the actin helix over the N-terminus of actin subdomain 1. The IgFLNa10 interaction appears to be specific, since no other individual Ig repeat or fragment of the FLNa molecule examined, besides ones with IgFLNa10 or CH domains, decorated F-actin filaments or were detected in reconstructions. We conclude that the combined interactions of CH domains and the IgFLNa10 repeat provide the binding strength of the whole FLNa molecule and propose a model for the association of IgFLNa10 on actin filaments.
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http://dx.doi.org/10.1016/j.jmb.2012.09.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3502691PMC
December 2012

Differential roles of cAMP and cGMP in megakaryocyte maturation and platelet biogenesis.

Exp Hematol 2013 Jan 11;41(1):91-101.e4. Epub 2012 Sep 11.

Institute of Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Würzburg, Germany.

The cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) regulate the activity of protein kinase A (PKA) and protein kinase G (PKG), respectively. This process helps maintain circulating platelets in a resting state. Here we studied the role of cAMP and cGMP in the regulation of megakaryocyte (MK) differentiation and platelet formation. Cultured, platelet-producing MKs were differentiated from fetal livers harvested from 13.5 days postcoital mouse embryos. MK development was accompanied by a dramatic increase in cAMP production and expression of soluble guanylate cyclase, PKG, and PKA as well as their downstream targets vasodilator-stimulated phosphoprotein (VASP) and MENA. Stimulation of prostaglandin E(1) receptor/adenylyl cyclase or soluble guanylate cyclase/PKG in cultured MKs increased VASP phosphorylation, indicating that these components share a common signaling pathway. To dissect out the role of cyclic nucleotides in MK differentiation, cAMP/PKA and cGMP/PKG signaling were alternately blocked in cultured MKs. Down-regulation of cAMP pathway effectors decreased MK numbers and ploidy. Notably, cGMP levels increased at the beginning of MK development and returned to basal levels in parallel with MK maturation. However, inhibition of cGMP pathway effectors had no effect on MK development. In addition, platelet release from mature MKs was enhanced by cGMP and inhibited by cAMP. Our data suggest that cAMP plays an important role in MK differentiation, while cAMP and cGMP have opposite effects on platelet production. Identifying the signaling pathways that underpin MK development and proplatelet formation will provide greater insights into thrombopoiesis and may potentially yield useful therapeutic targets.
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http://dx.doi.org/10.1016/j.exphem.2012.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3638753PMC
January 2013

Microtubule and cortical forces determine platelet size during vascular platelet production.

Nat Commun 2012 May 22;3:852. Epub 2012 May 22.

Hematology Division, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.

Megakaryocytes release large preplatelet intermediates into the sinusoidal blood vessels. Preplatelets convert into barbell-shaped proplatelets in vitro to undergo repeated abscissions that yield circulating platelets. These observations predict the presence of circular-preplatelets and barbell-proplatelets in blood, and two fundamental questions in platelet biology are what are the forces that determine barbell-proplatelet formation, and how is the final platelet size established. Here we provide insights into the terminal mechanisms of platelet production. We quantify circular-preplatelets and barbell-proplatelets in human blood in high-resolution fluorescence images, using a laser scanning cytometry assay. We demonstrate that force constraints resulting from cortical microtubule band diameter and thickness determine barbell-proplatelet formation. Finally, we provide a mathematical model for the preplatelet to barbell conversion. We conclude that platelet size is limited by microtubule bundling, elastic bending, and actin-myosin-spectrin cortex forces.
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http://dx.doi.org/10.1038/ncomms1838DOI Listing
May 2012

The origin and function of platelet glycosyltransferases.

Blood 2012 Jul 21;120(3):626-35. Epub 2012 May 21.

Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, Copenhagen N, Denmark.

Platelets are megakaryocyte subfragments that participate in hemostatic and host defense reactions and deliver pro- and antiangiogenic factors throughout the vascular system. Although they are anucleated cells that lack a complex secretory apparatus with distinct Golgi/endoplasmic reticulum compartments, past studies have shown that platelets have glycosyltransferase activities. In the present study, we show that members of 3 distinct glycosyltransferase families are found within and on the surface of platelets. Immunocytology and flow cytometry results indicated that megakaryocytes package these Golgi-derived glycosyltransferases into vesicles that are sent via proplatelets to nascent platelets, where they accumulate. These glycosyltransferases are active, and intact platelets glycosylate large exogenous substrates. Furthermore, we show that activation of platelets results in the release of soluble glycosyltransferase activities and that platelets contain sufficient levels of sugar nucleotides for detection of glycosylation of exogenously added substrates. Therefore, the results of the present study show that blood platelets are a rich source of both glycosyltransferases and donor sugar substrates that can be released to function in the extracellular space. This platelet-glycosylation machinery offers a pathway to a simple glycoengineering strategy improving storage of platelets and may serve hitherto unknown biologic functions.
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http://dx.doi.org/10.1182/blood-2012-02-409235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3401214PMC
July 2012

Extracellular DNA traps are associated with the pathogenesis of TRALI in humans and mice.

Blood 2012 Jun 17;119(26):6335-43. Epub 2012 May 17.

Immune Disease Institute and Program in Cellular and Molecular Medicine, Children's Hospital Boston, Boston, MA 02115, USA.

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related death. The biologic processes contributing to TRALI are poorly understood. All blood products can cause TRALI, and no specific treatment is available. A "2-event model" has been proposed as the trigger. The first event may include surgery, trauma, or infection; the second involves the transfusion of antileukocyte antibodies or bioactive lipids within the blood product. Together, these events induce neutrophil activation in the lungs, causing endothelial damage and capillary leakage. Neutrophils, in response to pathogens or under stress, can release their chromatin coated with granule contents, thus forming neutrophil extracellular traps (NETs). Although protective against infection, these NETs are injurious to tissue. Here we show that NET biomarkers are present in TRALI patients' blood and that NETs are produced in vitro by primed human neutrophils when challenged with anti-HNA-3a antibodies previously implicated in TRALI. NETs are found in alveoli of mice experiencing antibody-mediated TRALI. DNase 1 inhalation prevents their alveolar accumulation and improves arterial oxygen saturation even when administered 90 minutes after TRALI onset. We suggest that NETs form in the lungs during TRALI, contribute to the disease process, and thus could be targeted to prevent or treat TRALI.
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http://dx.doi.org/10.1182/blood-2012-01-405183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383196PMC
June 2012

Polymorphism in the protease-activated receptor-4 gene region associates with platelet activation and perioperative myocardial injury.

Am J Hematol 2012 Feb 7;87(2):161-6. Epub 2012 Jan 7.

Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.

Protease-activated receptors (PAR)-1 and -4 are the principal receptors for thrombin-mediated platelet activation. Functional genetic variation has been described in the human PAR1 gene, but not in the PAR4 gene (F2RL3). We sought to identify variants in and around F2RL3 and to determine their association with perioperative myocardial injury (PMI) after coronary artery bypass graft surgery. We further explored possible mechanisms for F2RL3 single nucleotide polymorphism (SNP) associations with PMI including altered receptor expression and platelet activation. Twenty-three SNPs in the F2RL3 gene region were genotyped in two phases in 934 Caucasian subjects. Platelets from 43 subjects (23 major allele, 20 risk allele) homozygous for rs773857 (SNP with the strongest association with PMI) underwent flow cytometry to assess PAR4 receptor number and response to activation by a specific PAR4 activating peptide (AYPGKF) measured by von Willebrand factor (vWf) binding and P-selectin release and PAC-1 binding. We identified a novel association of SNP rs773857 with PMI (OR = 2.4, P = 0.004). rs773857 risk allele homozygotes have significantly increased platelet counts and platelets showed a significant increase in P-selectin release after activation (P = 0.004). We conclude that rs773857 risk allele homozygotes are associated with risk for increased platelet count and hyperactivity.
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http://dx.doi.org/10.1002/ajh.22244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4371854PMC
February 2012

Desialylation accelerates platelet clearance after refrigeration and initiates GPIbα metalloproteinase-mediated cleavage in mice.

Blood 2012 Feb 18;119(5):1263-73. Epub 2011 Nov 18.

Translational Medicine Division, Brigham and Women's Hospital, Harvard Medical School, 1 Blackfan Circle, Boston, MA 02115, USA.

When refrigerated platelets are rewarmed, they secrete active sialidases, including the lysosomal sialidase Neu1, and express surface Neu3 that remove sialic acid from platelet von Willebrand factor receptor (VWFR), specifically the GPIbα subunit. The recovery and circulation of refrigerated platelets is greatly improved by storage in the presence of inhibitors of sialidases. Desialylated VWFR is also a target for metalloproteinases (MPs), because GPIbα and GPV are cleaved from the surface of refrigerated platelets. Receptor shedding is inhibited by the MP inhibitor GM6001 and does not occur in Adam17(ΔZn/ΔZn) platelets expressing inactive ADAM17. Critically, desialylation in the absence of MP-mediated receptor shedding is sufficient to cause the rapid clearance of platelets from circulation. Desialylation of platelet VWFR therefore triggers platelet clearance and primes GPIbα and GPV for MP-dependent cleavage.
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http://dx.doi.org/10.1182/blood-2011-05-355628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277358PMC
February 2012

Gelsolin-independent podosome formation in dendritic cells.

PLoS One 2011 11;6(7):e21615. Epub 2011 Jul 11.

Department of Medicine, Huddinge, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.

Podosomes, important structures for adhesion and extracellular matrix degradation, are claimed to be involved in cell migration. In addition, podosomes are also reported to be of importance in tissue remodelling, e.g., in osteoclast-mediated bone resorption. Podosomes are highly dynamic actin-filament scaffolds onto which proteins important for their function, such as matrix metallo-proteases and integrins, attach. The dynamics of the podosomes require the action of many proteins regulating actin assembly and disassembly. One such protein, gelsolin, which associates to podosomes, has been reported to be important for podosome formation and function in osteoclasts. However, podosome-like structures have been reported in gelsolin-deficient dendritic cells, but the identity of these structures was not confirmed, and their dynamics and function was not investigated. Like many other cells, dendritic cells of the immune system also form matrix degrading podosomes. In the present study, we show that dendritic cells form podosomes independently of gelsolin, that there are no major alterations in their dynamics of formation and disassembly, and that they exhibit matrix-degrading function. Furthermore, we found that gelsolin is not required for TLR4-induced podosome disassembly. Thus, the actin cytoskeleton of podosomes involved in dendritic cell extracellular matrix degradation appears to be regulated differently than the cytoskeleton in podosomes of osteoclasts mediating bone resorption.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021615PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3136926PMC
November 2011

RefilinB (FAM101B) targets filamin A to organize perinuclear actin networks and regulates nuclear shape.

Proc Natl Acad Sci U S A 2011 Jul 27;108(28):11464-9. Epub 2011 Jun 27.

Institut National de la Santé et de la Recherche Médicale, Unité 873, F-38054 Grenoble, France.

The intracellular localization and shape of the nucleus plays a central role in cellular and developmental processes. In fibroblasts, nuclear movement and shape are controlled by a specific perinuclear actin network made of contractile actin filament bundles called transmembrane actin-associated nuclear (TAN) lines that form a structure called the actin cap. The identification of regulatory proteins associated with this specific actin cytoskeletal dynamic is a priority for understanding actin-based changes in nuclear shape and position in normal and pathological situations. Here, we first identify a unique family of actin regulators, the refilin proteins (RefilinA and RefilinB), that stabilize specifically perinuclear actin filament bundles. We next identify the actin-binding filamin A (FLNA) protein as the downstream effector of refilins. Refilins act as molecular switches to convert FLNA from an actin branching protein into one that bundles. In NIH 3T3 fibroblasts, the RefilinB/FLNA complex organizes the perinuclear actin filament bundles forming the actin cap. Finally, we demonstrate that in epithelial normal murine mammary gland (NmuMG) cells, the RefilinB/FLNA complex controls formation of a new perinuclear actin network that accompanies nuclear shape changes during the epithelial-mesenchymal transition (EMT). Our studies open perspectives for further functional analyses of this unique actin-based network and shed light on FLNA function during development and in human syndromes associated with FLNA mutations.
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http://dx.doi.org/10.1073/pnas.1104211108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3136255PMC
July 2011

FlnA-null megakaryocytes prematurely release large and fragile platelets that circulate poorly.

Blood 2011 Aug 7;118(8):2285-95. Epub 2011 Jun 7.

Division of Translational Medicine, Brigham and Women's Hospital, and Department of Medicine, Harvard Medical School, Boston, MA, USA.

Filamin A (FlnA) is a large cytoplasmic protein that crosslinks actin filaments and anchors membrane receptors and signaling intermediates. FlnA(loxP) PF4-Cre mice that lack FlnA in the megakaryocyte (MK) lineage have a severe macrothrombocytopenia because of accelerated platelet clearance. Macrophage ablation by injection of clodronate-encapsulated liposomes increases blood platelet counts in FlnA(loxP) PF4-Cre mice and reveals the desintegration of FlnA-null platelets into microvesicles, a process that occurs spontaneously during storage. FlnA(loxP) PF4-Cre bone marrows and spleens have a 2.5- to 5-fold increase in MK numbers, indicating increased thrombopoiesis in vivo. Analysis of platelet production in vitro reveals that FlnA-null MKs prematurely convert their cytoplasm into large CD61(+) platelet-sized particles, reminiscent of the large platelets observed in vivo. FlnA stabilizes the platelet von Willebrand factor receptor, as surface expression of von Willebrand factor receptor components is normal on FlnA-null MKs but decreased on FlnA-null platelets. Further, FlnA-null platelets contain multiple GPIbα degradation products and have increased expression of the ADAM17 and MMP9 metalloproteinases. Together, the findings indicate that FlnA-null MKs prematurely release large and fragile platelets that are removed rapidly from the circulation by macrophages.
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http://dx.doi.org/10.1182/blood-2011-04-348482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3162356PMC
August 2011

The spectrin-based membrane skeleton stabilizes mouse megakaryocyte membrane systems and is essential for proplatelet and platelet formation.

Blood 2011 Aug 12;118(6):1641-52. Epub 2011 May 12.

Translational Medicine Division, Department of Medicine, Brigham & Women's Hospital, Boston, MA, USA.

Megakaryocytes generate platelets by remodeling their cytoplasm first into proplatelets and then into preplatelets, which undergo fission to generate platelets. Although the functions of microtubules and actin during platelet biogenesis have been defined, the role of the spectrin cytoskeleton is unknown. We investigated the function of the spectrin-based membrane skeleton in proplatelet and platelet production in murine megakaryocytes. Electron microscopy revealed that, like circulating platelets, proplatelets have a dense membrane skeleton, the main fibrous component of which is spectrin. Unlike other cells, megakaryocytes and their progeny express both erythroid and nonerythroid spectrins. Assembly of spectrin into tetramers is required for invaginated membrane system maturation and proplatelet extension, because expression of a spectrin tetramer-disrupting construct in megakaryocytes inhibits both processes. Incorporation of this spectrin-disrupting fragment into a novel permeabilized proplatelet system rapidly destabilizes proplatelets, causing blebbing and swelling. Spectrin tetramers also stabilize the "barbell shapes" of the penultimate stage in platelet production, because addition of the tetramer-disrupting construct converts these barbell shapes to spheres, demonstrating that membrane skeletal continuity maintains the elongated, pre-fission shape. The results of this study provide evidence for a role for spectrin in different steps of megakaryocyte development through its participation in the formation of invaginated membranes and in the maintenance of proplatelet structure.
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http://dx.doi.org/10.1182/blood-2011-01-330688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3156050PMC
August 2011

The filamins: organizers of cell structure and function.

Cell Adh Migr 2011 Mar-Apr;5(2):160-9. Epub 2011 Mar 1.

Translational Medicine Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.

Filamin A (FLNa), the first non-muscle actin filament cross-linking protein, was identified in 1975. Thirty five years of FLNa research has revealed its structure in great detail, discovered its isoforms (FLNb and c), and identified over 90 binding partners including channels, receptors, intracellular signaling molecules, and even transcription factors. Due to this diversity, mutations in human FLN genes result in a wide range of anomalies with moderate to lethal consequences. This review focuses on the structure and functions of FLNa in cell migration and adhesion.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084982PMC
http://dx.doi.org/10.4161/cam.5.2.14401DOI Listing
August 2011

Cell mechanics: Contracting to stiffness.

Nat Mater 2011 Jan;10(1):12-3

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http://dx.doi.org/10.1038/nmat2928DOI Listing
January 2011

Cytoskeletal mechanics of proplatelet maturation and platelet release.

J Cell Biol 2010 Nov;191(4):861-74

Translational Medicine Division, Brigham and Women's Hospital, Boston, MA 02115, USA.

Megakaryocytes generate platelets by remodeling their cytoplasm into long proplatelet extensions, which serve as assembly lines for platelet production. Although the mechanics of proplatelet elongation have been studied, the terminal steps of proplatelet maturation and platelet release remain poorly understood. To elucidate this process, released proplatelets were isolated, and their conversion into individual platelets was assessed. This enabled us to (a) define and quantify the different stages in platelet maturation, (b) identify a new intermediate stage in platelet production, the preplatelet, (c) delineate the cytoskeletal mechanics involved in preplatelet/proplatelet interconversion, and (d) model proplatelet fission and platelet release. Preplatelets are anucleate discoid particles 2-10 µm across that have the capacity to convert reversibly into elongated proplatelets by twisting microtubule-based forces that can be visualized in proplatelets expressing GFP-β1-tubulin. The release of platelets from the ends of proplatelets occurs at an increasing rate in time during culture, as larger proplatelets undergo successive fission, and is potentiated by shear.
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http://dx.doi.org/10.1083/jcb.201006102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2983072PMC
November 2010