Daniel F Cutler

University College London, Londinium, England, United Kingdom

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Publications (62)376.37 Total impact

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    ABSTRACT: Background: Many platelet functions are dependent on bioactive molecules released from their granules. Deficiencies of these granules in number, shape or content are associated with bleeding. The small size of these granules is such that imaging them for diagnosis has traditionally required electron microscopy. However, recently developed super-resolution microscopes provide sufficient spatial resolution to effectively image platelet granules. When combined with automated image analysis, these methods provide a quantitative, unbiased, rapidly acquired dataset that can readily and reliably reveal differences in platelet granules between individuals. Objective: To demonstrate the ability of structured illumination microscopy (SIM) to efficiently differentiate between healthy volunteers and three patients with Hermansky-Pudlak syndrome PATIENT METHODS: Blood samples were taken from three patients with Hermansky Pudlak syndrome and seven controls. Patients 1-3 have gene defects in HPS1, HPS6 and HPS5 respectively; all controls were healthy volunteers. Platelet-rich plasma was isolated from blood and the platelets fixed, stained for CD63, and processed for analysis by immunofluorescence microscopy, using a custom-built SIM microscope. Results: SIM can successfully resolve CD63-positive structures in fixed platelets. A determination of the number of CD63-positive structures per platelet allowed us to conclude that each patient was significantly different from all of the controls with 99% confidence. Conclusions: A super-resolution imaging approach is effective and rapid in objectively differentiating between patients with a platelet bleeding disorder and healthy volunteers. CD63 is a useful marker for predicting Hermansky-Pudlak Syndrome and could be used in the diagnosis of patients suspected of other Platelet granule disorders. This article is protected by copyright. All rights reserved.
    No preview · Article · Jan 2016 · Journal of Thrombosis and Haemostasis
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    ABSTRACT: Retinal degeneration and visual impairment are the first signs of juvenile neuronal ceroid lipofuscinosis caused by CLN3 mutations, followed by inevitable progression to blindness. We investigated retinal degeneration in Cln3Δex1-6 null mice, revealing classic ‘fingerprint’ lysosomal storage in the retinal pigment epithelium (RPE), replicating the human disease. The lysosomes contain mitochondrial F0-ATP synthase subunit c along with undigested membranes, indicating a reduced degradative capacity. Mature autophagosomes and basal phagolysosomes, the terminal degradative compartments of autophagy and phagocytosis, are also increased in Cln3Δex1-6 RPE, reflecting disruption to these key pathways that underpin the daily phagocytic turnover of photoreceptor outer segments (POS) required for maintenance of vision. The accumulated autophagosomes have post-lysosome fusion morphology, with undigested internal contents visible, while accumulated phagosomes are frequently docked to cathepsin D-positive lysosomes, without mixing of phagosomal and lysosomal contents. This suggests lysosome-processing defects affect both autophagy and phagocytosis, supported by evidence that phagosomes induced in Cln3Δex1-6-derived mouse embryonic fibroblasts have visibly disorganized membranes, unprocessed internal vesicles and membrane contents, in addition to reduced LAMP1 membrane recruitment. We propose that defective lysosomes in Cln3Δex1-6 RPE have a reduced degradative capacity that impairs the final steps of the intimately connected autophagic and phagocytic pathways that are responsible for degradation of POS. A build-up of degradative organellar by-products and decreased recycling of cellular materials is likely to disrupt processes vital to maintenance of vision by the RPE.
    Preview · Article · Oct 2015 · Human Molecular Genetics
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    ABSTRACT: Analysis of melanosome biogenesis in the retinal pigment epithelium (RPE) is challenging because it occurs predominantly in a short embryonic time window. Here we show that the zebrafish provides an ideal model system for studying this process because in the RPE the timing of melanosome biogenesis facilitates molecular manipulation using morpholinos. Morpholino-mediated knockdown of OA1, mutations in which cause the most common form of human ocular albinism, induces a major reduction in melanosome number, recapitulating a key feature of the mammalian disease where reduced melanosome numbers precede macromelanosome formation. We further show that PMEL, a key component of mammalian melanosome biogenesis, is required for generation of cylindrical melanosomes in zebrafish, in turn required for melanosome movement into the apical processes and maintenance of photoreceptor integrity. Spherical and cylindrical melanosomes containing similar melanin volumes co-exist in the cell body but only cylindrical melanosomes enter the apical processes. Taken together our findings indicate that melanosome number and shape are independently regulated and that melanosome shape controls a function in the RPE that depends on localization in the apical processes.
    Full-text · Article · Feb 2015 · Journal of Cell Science
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    ABSTRACT: Weibel-Palade bodies (WPBs), endothelial-specific secretory granules that are central to primary hemostasis and inflammation, occur in dimensions ranging between 0.5 and 5 μm. How their size is determined and whether it has a functional relevance are at present unknown. Here, we provide evidence for a dual role of the Golgi apparatus in controlling the size of these secretory carriers. At the ministack level, cisternae constrain the size of nanostructures ("quanta") of von Willebrand factor (vWF), the main WPB cargo. The ribbon architecture of the Golgi then allows copackaging of a variable number of vWF quanta within the continuous lumen of the trans-Golgi network, thereby generating organelles of different sizes. Reducing the WPB size abates endothelial cell hemostatic function by drastically diminishing platelet recruitment, but, strikingly, the inflammatory response (the endothelial capacity to engage leukocytes) is unaltered. Size can thus confer functional plasticity to an organelle by differentially affecting its activities.
    Full-text · Article · Apr 2014 · Developmental Cell
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    ABSTRACT: G protein-coupled receptors (GPCRs) are a major family of signaling molecules, central to the regulation of inflammatory responses. Their activation upon agonist binding is attenuated by GPCR kinases (GRKs), which desensitize the receptors through phosphorylation. G protein-coupled receptor kinase 2(GRK2) down-regulation in leukocytes has been closely linked to the progression of chronic inflammatory disorders such as rheumatoid arthritis and multiple sclerosis. Because leukocytes must interact with the endothelium to infiltrate inflamed tissues, we hypothesized that GRK2 down-regulation in endothelial cells would also be pro-inflammatory. To determine whether GRK2 down-regulation in endothelial cells is pro-inflammatory. siRNA-mediated ablation of GRK2 in human umbilical vein endothelial cells (HUVECs) was used in analyses of the role of this kinase. Microscopic and biochemical analyses of Weibel-Palade body (WPB) formation and functioning, live cell imaging of calcium concentrations and video analyses of adhesion of monocyte-like THP-1 cells provide clear evidence of GRK2 function in histamine activation of endothelial cells. G protein-coupled receptor kinase 2 depletion in HUVECs increases WPB exocytosis and P-selectin-dependent adhesion of THP-1 cells to the endothelial surface upon histamine stimulation, relative to controls. Further, live imaging of intracellular calcium concentrations reveals amplified histamine receptor signaling in GRK2-depleted cells, suggesting GRK2 moderates WPB exocytosis through receptor desensitization. G protein-coupled receptor kinase 2 deficiency in endothelial cells results in increased pro-inflammatory signaling and enhanced leukocyte recruitment to activated endothelial cells. The ability of GRK2 to modulate initiation of inflammatory responses in endothelial cells as well as leukocytes now places GRK2 at the apex of control of this finely balanced process.
    Full-text · Article · Feb 2014 · Journal of Thrombosis and Haemostasis
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    ABSTRACT: Figure optionsDownload full-size imageDownload high-quality image (295 K)Download as PowerPoint slide
    Full-text · Article · Jan 2014
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    T. Nightingale · D. Cutler
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    ABSTRACT: von Willebrand factor (VWF) plays key roles in both primary and secondary hemostasis by capturing platelets and chaperoning clotting factor VIII, respectively. It is stored within the Weibel–Palade bodies (WPBs) of endothelial cells as a highly prothrombotic protein, and its release is thus necessarily under tight control. Regulating the secretion of VWF involves multiple layers of cellular machinery that act together at different stages, leading to the exocytic fusion of WPBs with the plasma membrane and the consequent release of VWF. This review aims to provide a snapshot of the current understanding of those components, in particular the members of the Rab family, acting in the increasingly complex story of VWF secretion.
    Preview · Article · Jun 2013 · Journal of Thrombosis and Haemostasis
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    Full-text · Article · May 2013 · Heart (British Cardiac Society)
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    ABSTRACT: Von Willebrand disease (VWD) is a heterogeneous bleeding disorder caused by decrease or dysfunction of von Willebrand factor (VWF). A wide range of mutations in the VWF gene have been characterised; however their cellular consequences are still poorly understood. Here we have used a recently developed approach to study the molecular and cellular basis of VWD. We isolated blood outgrowth endothelial cells (BOEC) from peripheral blood of four type 1 VWD, four type 2 VWD patients and nine healthy controls. We confirmed the endothelial lineage of BOEC, then measured VWF mRNA and protein levels, both before and after stimulation, and VWF multimers. Decreased mRNA levels were predictive of plasma VWF levels in type 1 VWD, confirming a defect in VWF synthesis. However BOEC from this group of patients also showed defects in processing, storage and/or secretion of VWF. Levels of VWF mRNA and protein were normal in BOEC from three type 2 VWD patients, supporting the dysfunctional VWF model. However, one type 2M patient showed decreased VWF synthesis and storage, indicating a complex cellular defect. These results demonstrate for the first time that isolation of endothelial cells from VWD patients provides novel insight into the cellular mechanisms of the disease.
    Full-text · Article · Jan 2013 · Blood
  • Thomas D Nightingale · Daniel F Cutler · Louise P Cramer
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    ABSTRACT: It is well known that actin can associate with intracellular membranes to drive endocytosis and the rocketing motion of bacteria, virions and some organelles and to regulate synaptic vesicle plasticity. Actin also has been extensively reported to be involved at several steps of exocytosis; however, it has typically been described as functioning either within the actin cortex or by providing actin tracks for organelle transport. Increasingly, actin filament coats or rings have been directly localized on the surface of the exocytic organelle. Here, we suggest a common mechanism for actin-based regulation of large secretory granules whereby organelle-associated actomyosin II contraction either directly expels secretory content or stabilizes the exocytosing organelle.
    No preview · Article · Apr 2012 · Trends in cell biology
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    ABSTRACT: Dysregulation of angiogenesis is implicated in many diseases. Von Willebrand factor (VWF), a large plasma glycoprotein essential for normal haemostasis is synthesised by endothelial cells (EC) and megakaryocytes. Raised VWF plasma levels are a risk factor for arterial thrombosis, while deficiency of VWF causes Von Willebrand disease (VWD), the most common congenital bleeding disorder in man. VWD can be associated with angiodysplasia, vascular malformations linked to defective angiogenesis. We hypothesised that VWF is involved in angiogenesis. To test this hypothesis, we isolated mononuclear cells from peripheral blood of controls and patients with VWD and cultured them to obtain confluent monolayers of blood outgrowth endothelial cells (BOEC). BOEC from VWD patients showed decreased VWF release, consistent with the patients' clinical data, increased capillary tube formation on Matrigel, migration and proliferation compared to controls. Thus BOEC from VWD patients exhibit enhanced angiogenic properties. Increased angiogenesis was also observed after inhibition of VWF expression in human umbilical vein EC (HUVEC) with specific siRNA. Mechanism studies on VWF siRNA-treated HUVEC implicated the endothelial VWF receptor, integrin α V β3 and the angiogenesis regulator angiopoietin-2. To validate our findings in an in vivo model we studied the VWF-deficient mouse. In vivo Matrigel angiogenesis and imaging of blood vessels in the ear showed increased angiogenesis and vascular network compared to littermate controls. Thus we have identified a novel mechanism for the regulation of angiogenesis and a new function for VWF, which may have clinical implications for VWD and for cardiovascular disease.
    No preview · Article · Oct 2011 · Heart (British Cardiac Society)
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    ABSTRACT: The study of actin in regulated exocytosis has a long history with many different results in numerous systems. A major limitation on identifying precise mechanisms has been the paucity of experimental systems in which actin function has been directly assessed alongside granule content release at distinct steps of exocytosis of a single secretory organelle with sufficient spatiotemporal resolution. Using dual-color confocal microscopy and correlative electron microscopy in human endothelial cells, we visually distinguished two sequential steps of secretagogue-stimulated exocytosis: fusion of individual secretory granules (Weibel-Palade bodies [WPBs]) and subsequent expulsion of von Willebrand factor (VWF) content. Based on our observations, we conclude that for fusion, WPBs are released from cellular sites of actin anchorage. However, once fused, a dynamic ring of actin filaments and myosin II forms around the granule, and actomyosin II contractility squeezes VWF content out into the extracellular environment. This study therefore demonstrates how discrete actin cytoskeleton functions within a single cellular system explain actin filament-based prevention and promotion of specific exocytic steps during regulated secretion.
    Full-text · Article · Aug 2011 · The Journal of Cell Biology
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    ABSTRACT: The activation of endothelial cells is critical to initiating an inflammatory response. Activation induces the fusion of Weibel-Palade Bodies (WPB) with the plasma membrane, thus transferring P-selectin and VWF to the cell surface, where they act in the recruitment of leukocytes and platelets, respectively. CD63 has long been an established component of WPB, but the functional significance of its presence within an organelle that acts in inflammation and hemostasis was unknown. We find that ablating CD63 expression leads to a loss of P-selectin-dependent function: CD63-deficient HUVECs fail to recruit leukocytes, CD63-deficient mice exhibit a significant reduction in both leukocyte rolling and recruitment and we show a failure of leukocyte extravasation in a peritonitis model. Loss of CD63 has a similar phenotype to loss of P-selectin itself, thus CD63 is an essential cofactor to P-selectin.
    Full-text · Article · Jul 2011 · Blood
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    ABSTRACT: Von-Willebrand factor (vWF) is a highly multimerized hemostatic glycoprotein that is stored in endothelial Weibel-Palade bodies (WPB) and secreted upon cell stimulation to act in recruiting platelets to sites of vessel injury. Only fully matured multimeric vWF represents an efficient anchor for platelets, and endothelial cells have developed mechanisms to prevent release of immature vWF. Full maturation of vWF occurs within WPB following their translocation from a perinuclear site of emergence at the trans-Golgi network (TGN) to the cell periphery. The WPB-associated small GTPase Rab27a is involved in restricting immature WPB exocytosis and we searched for links between Rab27a and the actin cytoskeleton that could anchor WPB inside endothelial cells until they are fully matured. We here identify myosin Va as such link. Myosin Va forms a tripartite complex with Rab27a and its effector MyRIP and depletion of or dominant-negative interference with myosin Va leads to an increase in the ratio of perinuclear to more peripheral WPB. Concomitantly, myosin Va depletion results in an elevated secretion of less-oligomeric vWF from histamine-stimulated endothelial cells. These results indicate that a Rab27a/MyRIP/myosin Va complex is involved in linking WPB to the peripheral actin cytoskeleton of endothelial cells to allow full maturation and prevent premature secretion of vWF.
    Full-text · Article · Jul 2011 · Traffic
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    Full-text · Article · Mar 2011 · Blood
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    ABSTRACT: The regulation of blood vessel formation is of fundamental importance to many physiological processes, and angiogenesis is a major area for novel therapeutic approaches to diseases from ischemia to cancer. A poorly understood clinical manifestation of pathological angiogenesis is angiodysplasia, vascular malformations that cause severe gastrointestinal bleeding. Angiodysplasia can be associated with von Willebrand disease (VWD), the most common bleeding disorder in man. VWD is caused by a defect or deficiency in von Willebrand factor (VWF), a glycoprotein essential for normal hemostasis that is involved in inflammation. We hypothesized that VWF regulates angiogenesis. Inhibition of VWF expression by short interfering RNA (siRNA) in endothelial cells (ECs) caused increased in vitro angiogenesis and increased vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2)-dependent proliferation and migration, coupled to decreased integrin αvβ3 levels and increased angiopoietin (Ang)-2 release. ECs expanded from blood-derived endothelial progenitor cells of VWD patients confirmed these results. Finally, 2 different approaches, in situ and in vivo, showed increased vascularization in VWF-deficient mice. We therefore identify a new function of VWF in ECs, which confirms VWF as a protein with multiple vascular roles and defines a novel link between hemostasis and angiogenesis. These results may have important consequences for the management of VWD, with potential therapeutic implications for vascular diseases.
    No preview · Article · Nov 2010 · Blood
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    ABSTRACT: Endothelial von Willebrand factor (VWF) mediates platelet adhesion and acts as a protective chaperone to clotting factor VIII. Rapid release of highly multimerized VWF is particularly effective in promoting hemostasis. To produce this protein, an elaborate biogenesis is required, culminating at the trans-Golgi network (TGN) in storage within secretory granules called Weibel-Palade bodies (WPB). Failure to correctly form these organelles can lead to uncontrolled secretion of low-molecular-weight multimers of VWF. The TGN-associated adaptor AP-1 and its interactors clathrin, aftiphilin and γ-synergin are essential to initial WPB formation at the Golgi apparatus, and thus to VWF storage and secretion. To identify new proteins implicated in VWF storage and/or secretion. A genomewide RNA interference (RNAi) screen was performed in the Nematode C. elegans to identify new AP-1 genetic interactors. The small GTPase Rab10 was found to genetically interact with a partial loss of function of AP-1 in C. elegans. We investigated Rab10 in human primary umbilical vein endothelial cells (HUVECs). We report that Rab10 is enriched at the Golgi apparatus, where WPB are formed, and that in cells where Rab10 expression has been suppressed by siRNA, VWF secretion is altered: the amount of rapidly released VWF was significantly reduced. We also found that Rab8A has a similar function. Rab10 and Rab8A are new cytoplasmic factors implicated in WPB biogenesis that play a role in generating granules that can rapidly respond to secretagogue.
    Full-text · Article · Nov 2010 · Journal of Thrombosis and Haemostasis
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    ABSTRACT: Endothelial cells contain cigar-shaped secretory organelles called Weibel-Palade bodies (WPBs) that play a crucial role in both hemostasis and the initiation of inflammation. The major cargo protein of WPBs is von Willebrand factor (VWF). In unstimulated cells, this protein is stored in a highly multimerized state coiled into protein tubules, but after secretagogue stimulation and exocytosis it unfurls, under shear force, as long platelet-binding strings. Small GTPases of the Rab family play a key role in organelle function. Using siRNA depletion in primary endothelial cells, we have identified a role for the WPB-associated Rab27a and its effector MyRIP. Both these proteins are present on only mature WPBs, and this rab/effector complex appears to anchor these WPBs to peripheral actin. Depletion of either the Rab or its effector results in a loss of peripheral WPB localization, and this destabilization is coupled with an increase in both basal and stimulated secretion. The VWF released from Rab27a-depleted cells is less multimerized, and the VWF strings seen under flow are shorter. Our results indicate that this Rab/effector complex controls peripheral distribution and prevents release of incompletely processed WPB content.
    Full-text · Article · Apr 2009 · Blood
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    Daniel F Cutler
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    ABSTRACT: Platelets can be recruited by an ultra-large multimer fraction of von Willebrand factor extending from the endothelial surface into the plasma flow as long strings. In this issue of Blood, Huang and colleagues investigate the complex structure of these fishing lines and uncover a role for alpha(v)beta(3) integrin as their anchor to the endothelial surface.
    Preview · Article · Mar 2009 · Blood
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    ABSTRACT: Formation of secretory organelles requires the coupling of cargo selection to targeting into the correct exocytic pathway. Although the assembly of regulated secretory granules is driven in part by selective aggregation and retention of content, we recently reported that adaptor protein-1 (AP-1) recruitment of clathrin is essential to the initial formation of Weibel-Palade bodies (WPBs) at the trans-Golgi network. A selective co-aggregation process might include recruitment of components required for targeting to the regulated secretory pathway. However, we find that acquisition of the regulated secretory phenotype by WPBs in endothelial cells is coupled to but can be separated from formation of the distinctive granule core by ablation of the AP-1 effectors aftiphilin and gamma-synergin. Their depletion by small interfering RNA leads to WPBs that fail to respond to secretagogue and release their content in an unregulated manner. We find that these non-responsive WPBs have density, markers of maturation, and highly multimerized von Willebrand factor similar to those of wild-type granules. Thus, by also recruiting aftiphilin/gamma-synergin in addition to clathrin, AP-1 coordinates formation of WPBs with their acquisition of a regulated secretory phenotype.
    Full-text · Article · Oct 2008 · Molecular biology of the cell

Publication Stats

2k Citations
376.37 Total Impact Points

Institutions

  • 1994-2014
    • University College London
      • • MRC Laboratory for Molecular Cell Biology
      • • Department of Cell and Developmental Biology
      • • Department of Structural and Molecular Biology
      Londinium, England, United Kingdom
  • 2009
    • Imperial College London
      • Division of Cell and Molecular Biology
      Londinium, England, United Kingdom
  • 2002
    • Oregon Health and Science University
      Portland, Oregon, United States
  • 1999
    • University of Cambridge
      • Department of Biochemistry
      Cambridge, England, United Kingdom