Daniel F Cutler

University College London, Londinium, England, United Kingdom

Are you Daniel F Cutler?

Claim your profile

Publications (54)351.77 Total impact

  • Source
    [Show abstract] [Hide abstract]
    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.
    Developmental Cell 04/2014; · 12.86 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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.
    Journal of Thrombosis and Haemostasis 02/2014; 12(2):261-272. · 6.08 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Figure optionsDownload full-size imageDownload high-quality image (295 K)Download as PowerPoint slide
    01/2014;
  • Source
    T. Nightingale, D. Cutler
    [Show abstract] [Hide abstract]
    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.
    Journal of Thrombosis and Haemostasis 06/2013; 11(s1). · 6.08 Impact Factor
  • [Show abstract] [Hide abstract]
    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.
    Blood 01/2013; · 9.78 Impact Factor
  • [Show abstract] [Hide abstract]
    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.
    Trends in cell biology 04/2012; 22(6):329-37. · 12.12 Impact Factor
  • [Show abstract] [Hide abstract]
    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.
    Heart (British Cardiac Society) 10/2011; 97(20):e7. · 5.01 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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.
    The Journal of Cell Biology 08/2011; 194(4):613-29. · 10.82 Impact Factor
  • [Show abstract] [Hide abstract]
    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.
    Blood 07/2011; 118(15):4265-73. · 9.78 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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.
    Traffic 07/2011; 12(10):1371-82. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    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.
    Blood 11/2010; 117(3):1071-80. · 9.78 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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.
    Journal of Thrombosis and Haemostasis 11/2010; 9(2):392-401. · 6.08 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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.
    Blood 04/2009; 113(20):5010-8. · 9.78 Impact Factor
  • Source
    Daniel F Cutler
    [Show abstract] [Hide abstract]
    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.
    Blood 03/2009; 113(7):1397-8. · 9.78 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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.
    Molecular biology of the cell 10/2008; 19(12):5072-81. · 5.98 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are a group of inherited childhood-onset neurodegenerative disorders characterized by the lysosomal accumulation of undigested material within cells. To understand this dysfunction, we analysed trafficking of the cation-independent mannose 6-phosphate receptor (CI-MPR), which delivers the digestive enzymes to lysosomes. A common form of NCL is caused by mutations in CLN3, a multipass transmembrane protein of unknown function. We report that ablation of CLN3 causes accumulation of CI-MPR in the trans Golgi network, reflecting a 50% reduction in exit. This CI-MPR trafficking defect is accompanied by a fall in maturation and cellular activity of lysosomal cathepsins. CLN3 is therefore essential for trafficking along the route needed for delivery of lysosomal enzymes, and its loss thereby contributes to and may explain the lysosomal dysfunction underlying Batten disease.
    Traffic 10/2008; 9(11):1905-14. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Weibel-Palade bodies (WPBs) are secretory organelles used for post-synthesis storage in endothelial cells that can, very rapidly, be triggered to release their contents. They carry a variety of bioactive molecules that are needed to mount a rapid response to the complex environment of cells that line blood vessels. They store factors that are essential to haemostasis and inflammation, as well as factors that modulate vascular tonicity and angiogenesis. The number of WPBs and their precise content vary between endothelial tissues, reflecting their differing physiological circumstances. The particular functional demands of the highly multimerised haemostatic protein von Willebrand Factor (VWF), which is stored in WPBs as tubules until release, are responsible for the cigar shape of these granules. How VWF tubules drive the formation of these uniquely shaped organelles, and how WPB density increases during maturation, has recently been revealed by EM analysis using high-pressure freezing and freeze substitution. In addition, an AP1/clathrin coat has been found to be essential to WPB formation. Following recruitment of cargo at the TGN, there is a second wave of recruitment that delivers integral and peripheral membrane proteins to WPBs, some of which is AP3 dependent.
    Journal of Cell Science 02/2008; 121(Pt 1):19-27. · 5.88 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The neuronal ceroid lipofuscinoses (NCLs) are common neurodegenerative disorders of childhood and are classified as lysosomal storage diseases since affected cells exhibit lysosomes containing ceroid and lipofuscin-like material. CLN3 is the most widely conserved NCL gene, suggesting that it has a basic eukaryotic cell function; its loss might be expected to cause the earliest onset and/or most severe disease. However, mutations in CLN3 are linked to juvenile NCL (JNCL), the latest onset and mildest form of NCL in children. We sought to explain this paradox. Almost all patients with JNCL are homozygous or heterozygous for an intragenic 1 kb deletion within CLN3, hitherto presumed to be a null mutation. We hypothesized that the 1 kb mutation may allow CLN3 residual function. We confirmed the presence of CLN3 transcripts in JNCL patient cells. When RNA silencing was used to deplete these transcripts in cells from JNCL patients, the lysosomes significantly increased in size, confirming the presence of functional protein in these cells. Consistently, overexpression of mutant CLN3 transcript caused lysosomes to decrease in size. We modelled the JNCL mutant transcripts and those corresponding to mouse models for Cln3 in Schizosaccharomyces pombe and confirmed that most transcripts retained significant function as we predicted. Therefore, we concluded that the common mutant CLN3 protein does indeed retain significant function and that JNCL is a mutation-specific disease phenotype. This finding has important consequences for recognition and diagnosis of disease caused by mutations in CLN3 and for the development of therapy for JNCL.
    Human Molecular Genetics 02/2008; 17(2):303-12. · 7.69 Impact Factor
  • Source
    Graça Raposo, Michael S Marks, Daniel F Cutler
    [Show abstract] [Hide abstract]
    ABSTRACT: Some cells harbour specialised lysosome-related organelles (LROs) that share features of late endosomes/lysosomes but are functionally, morphologically and/or compositionally distinct. Ubiquitous trafficking machineries cooperate with cell type specific cargoes to produce these organelles. Several genetic diseases are caused by dysfunctional LRO formation and/or motility. Many genes affected by these diseases have been recently identified, revealing new cellular components of the trafficking machinery. Current research reveals how the products of these genes cooperate to generate LROs and how these otherwise diverse organelles are related by the mechanisms through which they form.
    Current Opinion in Cell Biology 09/2007; 19(4):394-401. · 11.41 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The Weibel-Palade bodies (WPBs) of endothelial cells play an important role in haemostasis and the initiation of inflammation, yet their biogenesis is poorly understood. Tubulation of their major content protein, von Willebrand factor (VWF), is crucial to WPB function, and so we investigated further the relationship between VWF tubule formation and WPB formation in human umbilical vein endothelial cells (HUVECs). By using high-pressure freezing and freeze substitution before electron microscopy, we visualised VWF tubules in the trans-Golgi network (TGN), as well as VWF subunits in vesicular structures. Tubules were also seen in WPBs that were connected to the TGN by membranous stalks. Tubules are disorganised in the immature WPBs but during maturation we found a dramatic increase in the spatial organisation of the tubules and in organelle electron density. We also found coated budding profiles suggestive of the removal of missorted material after initial formation of these granules. Finally, we discovered that these large, seemingly rigid, organelles flex at hinge points and that the VWF tubules are interrupted at these hinges, facilitating organelle movement around the cell. The use of high-pressure freezing was vital in this study and it suggests that this technique might prove essential to any detailed characterisation of organelle biogenesis.
    Journal of Cell Science 07/2007; 120(Pt 12):2117-25. · 5.88 Impact Factor

Publication Stats

2k Citations
351.77 Total Impact Points

Institutions

  • 1994–2014
    • University College London
      • MRC Laboratory for Molecular Cell Biology
      Londinium, England, United Kingdom
  • 2010
    • Université de Rennes 1
      Roazhon, Brittany, France
  • 2007
    • Institut Curie
      Lutetia Parisorum, Île-de-France, France
  • 2002
    • Oregon Health and Science University
      Portland, Oregon, United States
  • 1989
    • Imperial College London
      Londinium, England, United Kingdom