Alan Prescott - Academia.edu (original) (raw)

Papers by Alan Prescott

Nature, Apr 1, 2002

at the rim of the gyre, where the parent originating from return Atlantic water and that coming f... more at the rim of the gyre, where the parent originating from return Atlantic water and that coming from inside-gyre surface waters, meet and mix. Because these coherent eddies are stable for ,1 year, they may also precondition water masses for convective activity in the following winter season. They could then form foci to concentrate further convection 4,11,12 after erosion of the layer of less dense water that caps the core during the summer. On the basis of float data only, we have direct evidence for about eight such eddies during ESOP-2. This probably underestimates the true number, because almost every float we released that strayed into the rim region of the Greenland gyre in 1997 became entrained in an anticyclonic eddy. When these eddies eventually decay, they presumably release their core water at mid-depths in the Greenland Sea, ventilating the intermediate water. Each eddy core has a volume ,50 km 3. Comparing the volume of water in eight eddy cores to a total volume involved in convection of (2-4) £ 10 12 m 3 during 1996-97, a figure we have previously calculated from analysis of the tracer release experiment 7 , suggests a contribution of 10-20% to the total amount of convection from the eddies. However, only a small proportion-less than 10% of the total amount of water involved in convection-penetrated to around 1,000 m or deeper, most being confined to the upper ,500 m. Thus the eddies made a significant contribution to the total volume of deep convectionand dominated the water injected to substantial depth-in the Greenland Sea in 1996-97. Greenland Arctic Intermediate Water is thought to contribute to the overflows leading into the North Atlantic deep water, and the eddies thus provide a pathway for ventilation of the deep North Atlantic. Long-lived submesoscale anticyclonic vortices have also been observed in the Labrador Sea 13,14 (another site known for deep ocean convection), indicating that such vortices may be ubiquitous features of deep ocean convection.

Mechanisms of Development, Aug 1, 2009

Journal of Molecular Cell Biology, Jun 10, 2015

Dendritic cells (DC) are the major antigen-presenting cells bridging innate and adaptive immunity... more Dendritic cells (DC) are the major antigen-presenting cells bridging innate and adaptive immunity, a function they perform by converting quiescent DC to active, mature DC with the capacity to activate naïve T cells. They do this by migrating from the tissues to the T cell area of the secondary lymphoid tissues. Here, we demonstrate that myeloid cell-specific genetic deletion of PTP1B (LysM PTP1B) leads to defects in lipopolysaccharide-driven bone marrow-derived DC (BMDC) activation associated with increased levels of phosphorylated Stat3. We show that myeloid cell-specific PTP1B deletion also causes decreased migratory capacity of epidermal DC, as well as reduced CCR7 expression and chemotaxis to CCL19 by BMDC. PTP1B deficiency in BMDC also impairs their migration in vivo. Further, immature LysM PTP1B BMDC display fewer podosomes, increased levels of phosphorylated Src at tyrosine 527, and loss of Src localization to podosome puncta. In co-culture with T cells, LysM PTP1B BMDC establish fewer and shorter contacts than control BMDC. Finally, LysM PTP1B BMDC fail to present antigen to T cells as efficiently as control BMDC. These data provide first evidence for a key regulatory role for PTP1B in mediating a central DC function of initiating adaptive immune responses in response to innate immune cell activation.

PLOS ONE, Jul 16, 2012

Electrical gradients are present in many developing and regenerating tissues and around tumours. ... more Electrical gradients are present in many developing and regenerating tissues and around tumours. Mimicking endogenous electric fields in vitro has profound effects on the behaviour of many cell types. Intriguingly, specific cell types migrate cathodally, others anodally and some polarise with their long axis perpendicular to the electric vector. These striking phenomena are likely to have in vivo relevance since one of the determining factors during cancer metastasis is the ability to switch between attractive and repulsive migration in response to extracellular guidance stimuli. We present evidence that the cervical cancer cell line HeLa migrates cathodally in a direct current electric field of physiological intensity, while the strongly metastatic prostate cancer cell line PC-3-M migrates anodally. Notably, genetic disruption of protein serine/ threonine phosphatase-1 (PP1) and its regulator NIPP1 decrease directional migration in these cell lines. Conversely, the inducible expression of NIPP1 switched the directional response of HeLa cells from cathodal to slightly anodal in a PP1dependent manner. Remarkably, induction of a hyperactive PP1/NIPP1 holoenzyme, further shifted directional migration towards the anode. We show that PP1 association with NIPP1 upregulates signalling by the GTPase Cdc42 and demonstrate that pharmacological inhibition of Cdc42 in cells overexpressing NIPP1 recovered cathodal migration. Taken together, we provide the first evidence for regulation of directional cell migration by NIPP1. In addition, we identify PP1/NIPP1 as a novel molecular compass that controls directed cell migration via upregulation of Cdc42 signalling and suggest a way by which PP1/NIPP1 may contribute to the migratory properties of cancer cells.

Molecular Biology of the Cell, 2003

Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase that regulates many cell... more Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase that regulates many cellular processes, including cell division. When transiently expressed as fluorescent protein (FP) fusions, the three PP1 isoforms, ␣, ␤/␦, and ␥1, are active phosphatases with distinct localization patterns. We report here the establishment and characterization of HeLa cell lines stably expressing either FP-PP1␥ or FP alone. Time-lapse imaging reveals dynamic targeting of FP-PP1␥ to specific sites throughout the cell cycle, contrasting with the diffuse pattern observed for FP alone. FP-PP1␥ shows a nucleolar accumulation during interphase. On entry into mitosis, it localizes initially at kinetochores, where it exchanges rapidly with the diffuse cytoplasmic pool. A dramatic relocalization of PP1 to the chromosome-containing regions occurs at the transition from early to late anaphase, and by telophase FP-PP1␥ also accumulates at the cleavage furrow and midbody. The changing spatio-temporal distribution of PP1␥ revealed using the stable PP1 cell lines implicates it in multiple processes, including nucleolar function, the regulation of chromosome segregation and cytokinesis.

The International Journal of Biochemistry & Cell Biology, 2008

The mechanisms that coordinate centrosome maturation and the migration of human cells remain elus... more The mechanisms that coordinate centrosome maturation and the migration of human cells remain elusive. Protein phosphatase 4 (Ppp4) is a ubiquitous protein serine/threonine phosphatase in eukaryotes that is enriched at centrosomes. HEK293 cells cultures depleted to 30% Ppp4c levels by lentivirus-delivered stable gene silencing were delayed in mitosis at the prometaphase/metaphase boundary and displayed cells with aberrant chromosome organisation and microtubules unconnected to the centrosomes. The levels of ␣and ␥-tubulin and aurora A were decreased; in mitotic cells, the cytological localisations of polo-like kinase 1, ␣and ␥-tubulin and aurora A were aberrant and the phosphorylation of Aurora A-Thr 288 was decreased. The novel localisation of endogenous Ppp4 regulatory subunit, R3A, to centrosomes in human mitotic cells suggests that a Ppp4c-R2-R3 trimeric complex mediates centrosome maturation. We demonstrate for the first time that human cells depleted to 30% Ppp4c showed severely decreased migration and exhibit decreased levels of both total ␤-actin and filamentous actin in cell extensions, filopodia and lamellopodia-like structures. Our studies show that Ppp4c is required for the organisation of the actin cytoskeleton at the leading edge of human cells during migration. We also demonstrate that the active forms of the RhoGTPases, Rac1 and Cdc42, are substantially decreased in the presence and absence of growth factor in Ppp4c depleted cells, implicating Ppp4c in the regulation of these GTPases. The results suggest that Ppp4c-R2-R3 complexes may coordinate centrosome maturation and cell migration via regulation of RhoGTPases and that Ppp4 may be a useful anticancer target.

Development, Feb 15, 2009

The chicken talpid 3 mutant, with polydactyly and defects in other embryonic regions that depend ... more The chicken talpid 3 mutant, with polydactyly and defects in other embryonic regions that depend on hedgehog (Hh) signalling (e.g. the neural tube), has a mutation in KIAA0568. Similar phenotypes are seen in mice and in human syndromes with mutations in genes that encode centrosomal or intraflagella transport proteins. Such mutations lead to defects in primary cilia, sites where Hh signalling occurs. Here, we show that cells of talpid 3 mutant embryos lack primary cilia and that primary cilia can be rescued with constructs encoding Talpid3. talpid 3 mutant embryos also develop polycystic kidneys, consistent with widespread failure of ciliogenesis. Ultrastructural studies of talpid 3 mutant neural tube show that basal bodies mature but fail to dock with the apical cell membrane, are misorientated and almost completely lack ciliary axonemes. We also detected marked changes in actin organisation in talpid 3 mutant cells, which may explain misorientation of basal bodies. KIAA0586 was identified in the human centrosomal proteome and, using an antibody against chicken Talpid3, we detected Talpid3 in the centrosome of wild-type chicken cells but not in mutant cells. Cloning and bioinformatic analysis of the Talpid3 homolog from the sea anemone Nematostella vectensis identified a highly conserved region in the Talpid3 protein, including a predicted coiled-coil domain. We show that this region is required to rescue primary cilia formation and neural tube patterning in talpid 3 mutant embryos, and is sufficient for centrosomal localisation. Thus, Talpid3 is one of a growing number of centrosomal proteins that affect both ciliogenesis and Hh signalling.

Molecular Biology of the Cell, Nov 1, 2007

The lens is an avascular tissue, separated from the aqueous and vitreous humors by its own extrac... more The lens is an avascular tissue, separated from the aqueous and vitreous humors by its own extracellular matrix, the lens capsule. Here we demonstrate that the lens capsule is a source of essential survival factors for lens epithelial cells. Primary and immortalized lens epithelial cells survive in low levels of serum and are resistant to staurosporine-induced apoptosis when they remain in contact with the lens capsule. Physical contact with the capsule is required for maximal resistance to stress. The lens capsule is also a source of soluble factors including fibroblast growth factor 2 (FGF-2) and perlecan, an extracellular matrix component that enhances FGF-2 activity. Matrix metalloproteinase 2 (MMP-2) inhibition as well as MMP-2 pretreatment of lens capsules greatly reduced the protective effect of the lens capsule, although this could be largely reversed by the addition of either conditioned medium or recombinant FGF-2. These data suggest that FGF-2 release from the lens capsule by MMP-2 is essential to lens epithelial cell viability and survival.

Journal of Cell Science, Mar 10, 2022

Investigative Ophthalmology & Visual Science, 2005

Investigative Ophthalmology & Visual Science, 2002

Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell cu... more Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and <i>in vitro</i> studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation <i>in vivo</i> in mammals remains unknown. To address this, we generated a <i>Parkin</i> Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in <i>Parkin</i>^S65A/S65A neurons. Phenotypically, <i>Parkin</i>^S65A/S65A mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (<i>PARK2</i>) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the Parkin^S65N/S65N mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.

Dynamic contacts between cells within the developing neuroepithelium are poorly understood but pl... more Dynamic contacts between cells within the developing neuroepithelium are poorly understood but play important roles in cell and tissue morphology and cell signalling. Here, using live-cell imaging and electron microscopy we reveal multiple protrusive structures in neuroepithelial apical endfeet of the chick embryonic spinal cord, including sub-apical protrusions that extend laterally within the tissue, and observe similar structures in human neuroepithelium. We characterise the dynamics, shape, and cytoskeleton of these lateral protrusions and distinguish these structures from cytonemes/filopodia and tunnelling nanotubes. We demonstrate that lateral protrusions form a latticework of membrane contacts between non-adjacent cells, depend on actin but not microtubule dynamics and provide a lamellipodial-like platform for further extending fine actin-dependent filipodia. We find that lateral protrusions depend on the actin-binding protein WAVE1: mutant-WAVE1 misexpression attenuated prot...

Investigative Ophthalmology & Visual Science, 2003

Wellcome Open Research, 2019

Background:Atopic eczema is an itchy inflammatory disorder characterised by skin barrier dysfunct... more Background:Atopic eczema is an itchy inflammatory disorder characterised by skin barrier dysfunction. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a major risk factor, but the mechanisms by which filaggrin haploinsufficiency leads to atopic inflammation remain incompletely understood. Skin as an organ that can be modelled using primary cellsin vitroprovides the opportunity for selected genetic effects to be investigated in detail.Methods:Primary human keratinocytes and donor-matched primary fibroblasts from healthy individuals were used to create skin organoid models with and without siRNA-mediated knockdown ofFLG. Biological replicate sets of organoids were assessed using histological, functional and biochemical measurements.Results:FLGknockdown leads to subtle changes in histology and ultrastructure including a reduction in thickness of the stratum corneum and smaller, less numerous keratohyalin granules. Immature organoids showed some limited evidence of ba...

Mutations enhancing the kinase activity of LRRK2 cause Parkinson’s disease (PD) and therapies tha... more Mutations enhancing the kinase activity of LRRK2 cause Parkinson’s disease (PD) and therapies that reduce LRRK2 kinase activity are being tested in clinical trials. Numerous rare variants of unknown clinical significance have been reported, but how the vast majority impact on LRRK2 function is unknown. Here, we investigate 100 LRRK2 variants linked to PD, including previously described pathogenic mutations. We identify 23 LRRK2 variants that robustly stimulate kinase activity, including variants within the N-terminal non-catalytic regions [ARM (E334K, A419V), ANK(R767H), LRR (R1067Q, R1325Q)], as well as variants predicted to destabilise the ROC:CORB interface [ROC (A1442P, V1447M), CORA (R1628P) CORB (S1761R, L1795F)] and COR:COR dimer interface [CORB (R1728H/L)]. Most activating variants decrease LRRK2 biomarker site phosphorylation (pSer935/pSer955/pSer973), consistent with the notion that the active kinase conformation blocks their phosphorylation. We conclude that the impact of...

www.embojournal.org Structural insights into the regulation of PDK1 by phosphoinositides and inos... more www.embojournal.org Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates

After 2 h at 37°C, migrated or input cells were recovered from the wells, quantitated by flow cyt... more After 2 h at 37°C, migrated or input cells were recovered from the wells, quantitated by flow cytometry, and expressed as ratios of migrated or input HeN/HeJ. (B) DC were pretreated with 100 ng/ml LPS for different lengths of time before addition into the Transwell insert. Note that time = 0* data were obtained from cells where LPS was added immediately before the cells were placed onto the filter. Subsequent migration was for 2 h in all cases. The line indicates the 1:1 ratio achieved where HeN and HeJ migrate with equal efficiency. (C) Data from experiments on three or four independent DC cultures are shown. Bars represent mean data. *, P < 0.05.<b>Copyright information:</b>Taken from "TLR ligand–induced podosome disassembly in dendritic cells is ADAM17 dependent"The Journal of Cell Biology 2008;182(5):993-1005.Published online 8 Sep 2008PMCID:PMC2528573.

Investigative Ophthalmology & Visual Science, 2005

Nature, Apr 1, 2002

at the rim of the gyre, where the parent originating from return Atlantic water and that coming f... more at the rim of the gyre, where the parent originating from return Atlantic water and that coming from inside-gyre surface waters, meet and mix. Because these coherent eddies are stable for ,1 year, they may also precondition water masses for convective activity in the following winter season. They could then form foci to concentrate further convection 4,11,12 after erosion of the layer of less dense water that caps the core during the summer. On the basis of float data only, we have direct evidence for about eight such eddies during ESOP-2. This probably underestimates the true number, because almost every float we released that strayed into the rim region of the Greenland gyre in 1997 became entrained in an anticyclonic eddy. When these eddies eventually decay, they presumably release their core water at mid-depths in the Greenland Sea, ventilating the intermediate water. Each eddy core has a volume ,50 km 3. Comparing the volume of water in eight eddy cores to a total volume involved in convection of (2-4) £ 10 12 m 3 during 1996-97, a figure we have previously calculated from analysis of the tracer release experiment 7 , suggests a contribution of 10-20% to the total amount of convection from the eddies. However, only a small proportion-less than 10% of the total amount of water involved in convection-penetrated to around 1,000 m or deeper, most being confined to the upper ,500 m. Thus the eddies made a significant contribution to the total volume of deep convectionand dominated the water injected to substantial depth-in the Greenland Sea in 1996-97. Greenland Arctic Intermediate Water is thought to contribute to the overflows leading into the North Atlantic deep water, and the eddies thus provide a pathway for ventilation of the deep North Atlantic. Long-lived submesoscale anticyclonic vortices have also been observed in the Labrador Sea 13,14 (another site known for deep ocean convection), indicating that such vortices may be ubiquitous features of deep ocean convection.

Mechanisms of Development, Aug 1, 2009

Journal of Molecular Cell Biology, Jun 10, 2015

Dendritic cells (DC) are the major antigen-presenting cells bridging innate and adaptive immunity... more Dendritic cells (DC) are the major antigen-presenting cells bridging innate and adaptive immunity, a function they perform by converting quiescent DC to active, mature DC with the capacity to activate naïve T cells. They do this by migrating from the tissues to the T cell area of the secondary lymphoid tissues. Here, we demonstrate that myeloid cell-specific genetic deletion of PTP1B (LysM PTP1B) leads to defects in lipopolysaccharide-driven bone marrow-derived DC (BMDC) activation associated with increased levels of phosphorylated Stat3. We show that myeloid cell-specific PTP1B deletion also causes decreased migratory capacity of epidermal DC, as well as reduced CCR7 expression and chemotaxis to CCL19 by BMDC. PTP1B deficiency in BMDC also impairs their migration in vivo. Further, immature LysM PTP1B BMDC display fewer podosomes, increased levels of phosphorylated Src at tyrosine 527, and loss of Src localization to podosome puncta. In co-culture with T cells, LysM PTP1B BMDC establish fewer and shorter contacts than control BMDC. Finally, LysM PTP1B BMDC fail to present antigen to T cells as efficiently as control BMDC. These data provide first evidence for a key regulatory role for PTP1B in mediating a central DC function of initiating adaptive immune responses in response to innate immune cell activation.

PLOS ONE, Jul 16, 2012

Electrical gradients are present in many developing and regenerating tissues and around tumours. ... more Electrical gradients are present in many developing and regenerating tissues and around tumours. Mimicking endogenous electric fields in vitro has profound effects on the behaviour of many cell types. Intriguingly, specific cell types migrate cathodally, others anodally and some polarise with their long axis perpendicular to the electric vector. These striking phenomena are likely to have in vivo relevance since one of the determining factors during cancer metastasis is the ability to switch between attractive and repulsive migration in response to extracellular guidance stimuli. We present evidence that the cervical cancer cell line HeLa migrates cathodally in a direct current electric field of physiological intensity, while the strongly metastatic prostate cancer cell line PC-3-M migrates anodally. Notably, genetic disruption of protein serine/ threonine phosphatase-1 (PP1) and its regulator NIPP1 decrease directional migration in these cell lines. Conversely, the inducible expression of NIPP1 switched the directional response of HeLa cells from cathodal to slightly anodal in a PP1dependent manner. Remarkably, induction of a hyperactive PP1/NIPP1 holoenzyme, further shifted directional migration towards the anode. We show that PP1 association with NIPP1 upregulates signalling by the GTPase Cdc42 and demonstrate that pharmacological inhibition of Cdc42 in cells overexpressing NIPP1 recovered cathodal migration. Taken together, we provide the first evidence for regulation of directional cell migration by NIPP1. In addition, we identify PP1/NIPP1 as a novel molecular compass that controls directed cell migration via upregulation of Cdc42 signalling and suggest a way by which PP1/NIPP1 may contribute to the migratory properties of cancer cells.

Molecular Biology of the Cell, 2003

Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase that regulates many cell... more Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase that regulates many cellular processes, including cell division. When transiently expressed as fluorescent protein (FP) fusions, the three PP1 isoforms, ␣, ␤/␦, and ␥1, are active phosphatases with distinct localization patterns. We report here the establishment and characterization of HeLa cell lines stably expressing either FP-PP1␥ or FP alone. Time-lapse imaging reveals dynamic targeting of FP-PP1␥ to specific sites throughout the cell cycle, contrasting with the diffuse pattern observed for FP alone. FP-PP1␥ shows a nucleolar accumulation during interphase. On entry into mitosis, it localizes initially at kinetochores, where it exchanges rapidly with the diffuse cytoplasmic pool. A dramatic relocalization of PP1 to the chromosome-containing regions occurs at the transition from early to late anaphase, and by telophase FP-PP1␥ also accumulates at the cleavage furrow and midbody. The changing spatio-temporal distribution of PP1␥ revealed using the stable PP1 cell lines implicates it in multiple processes, including nucleolar function, the regulation of chromosome segregation and cytokinesis.

The International Journal of Biochemistry & Cell Biology, 2008

The mechanisms that coordinate centrosome maturation and the migration of human cells remain elus... more The mechanisms that coordinate centrosome maturation and the migration of human cells remain elusive. Protein phosphatase 4 (Ppp4) is a ubiquitous protein serine/threonine phosphatase in eukaryotes that is enriched at centrosomes. HEK293 cells cultures depleted to 30% Ppp4c levels by lentivirus-delivered stable gene silencing were delayed in mitosis at the prometaphase/metaphase boundary and displayed cells with aberrant chromosome organisation and microtubules unconnected to the centrosomes. The levels of ␣and ␥-tubulin and aurora A were decreased; in mitotic cells, the cytological localisations of polo-like kinase 1, ␣and ␥-tubulin and aurora A were aberrant and the phosphorylation of Aurora A-Thr 288 was decreased. The novel localisation of endogenous Ppp4 regulatory subunit, R3A, to centrosomes in human mitotic cells suggests that a Ppp4c-R2-R3 trimeric complex mediates centrosome maturation. We demonstrate for the first time that human cells depleted to 30% Ppp4c showed severely decreased migration and exhibit decreased levels of both total ␤-actin and filamentous actin in cell extensions, filopodia and lamellopodia-like structures. Our studies show that Ppp4c is required for the organisation of the actin cytoskeleton at the leading edge of human cells during migration. We also demonstrate that the active forms of the RhoGTPases, Rac1 and Cdc42, are substantially decreased in the presence and absence of growth factor in Ppp4c depleted cells, implicating Ppp4c in the regulation of these GTPases. The results suggest that Ppp4c-R2-R3 complexes may coordinate centrosome maturation and cell migration via regulation of RhoGTPases and that Ppp4 may be a useful anticancer target.

Development, Feb 15, 2009

The chicken talpid 3 mutant, with polydactyly and defects in other embryonic regions that depend ... more The chicken talpid 3 mutant, with polydactyly and defects in other embryonic regions that depend on hedgehog (Hh) signalling (e.g. the neural tube), has a mutation in KIAA0568. Similar phenotypes are seen in mice and in human syndromes with mutations in genes that encode centrosomal or intraflagella transport proteins. Such mutations lead to defects in primary cilia, sites where Hh signalling occurs. Here, we show that cells of talpid 3 mutant embryos lack primary cilia and that primary cilia can be rescued with constructs encoding Talpid3. talpid 3 mutant embryos also develop polycystic kidneys, consistent with widespread failure of ciliogenesis. Ultrastructural studies of talpid 3 mutant neural tube show that basal bodies mature but fail to dock with the apical cell membrane, are misorientated and almost completely lack ciliary axonemes. We also detected marked changes in actin organisation in talpid 3 mutant cells, which may explain misorientation of basal bodies. KIAA0586 was identified in the human centrosomal proteome and, using an antibody against chicken Talpid3, we detected Talpid3 in the centrosome of wild-type chicken cells but not in mutant cells. Cloning and bioinformatic analysis of the Talpid3 homolog from the sea anemone Nematostella vectensis identified a highly conserved region in the Talpid3 protein, including a predicted coiled-coil domain. We show that this region is required to rescue primary cilia formation and neural tube patterning in talpid 3 mutant embryos, and is sufficient for centrosomal localisation. Thus, Talpid3 is one of a growing number of centrosomal proteins that affect both ciliogenesis and Hh signalling.

Molecular Biology of the Cell, Nov 1, 2007

The lens is an avascular tissue, separated from the aqueous and vitreous humors by its own extrac... more The lens is an avascular tissue, separated from the aqueous and vitreous humors by its own extracellular matrix, the lens capsule. Here we demonstrate that the lens capsule is a source of essential survival factors for lens epithelial cells. Primary and immortalized lens epithelial cells survive in low levels of serum and are resistant to staurosporine-induced apoptosis when they remain in contact with the lens capsule. Physical contact with the capsule is required for maximal resistance to stress. The lens capsule is also a source of soluble factors including fibroblast growth factor 2 (FGF-2) and perlecan, an extracellular matrix component that enhances FGF-2 activity. Matrix metalloproteinase 2 (MMP-2) inhibition as well as MMP-2 pretreatment of lens capsules greatly reduced the protective effect of the lens capsule, although this could be largely reversed by the addition of either conditioned medium or recombinant FGF-2. These data suggest that FGF-2 release from the lens capsule by MMP-2 is essential to lens epithelial cell viability and survival.

Journal of Cell Science, Mar 10, 2022

Investigative Ophthalmology & Visual Science, 2005

Investigative Ophthalmology & Visual Science, 2002

Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell cu... more Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and <i>in vitro</i> studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation <i>in vivo</i> in mammals remains unknown. To address this, we generated a <i>Parkin</i> Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in <i>Parkin</i>^S65A/S65A neurons. Phenotypically, <i>Parkin</i>^S65A/S65A mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (<i>PARK2</i>) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the Parkin^S65N/S65N mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.

Dynamic contacts between cells within the developing neuroepithelium are poorly understood but pl... more Dynamic contacts between cells within the developing neuroepithelium are poorly understood but play important roles in cell and tissue morphology and cell signalling. Here, using live-cell imaging and electron microscopy we reveal multiple protrusive structures in neuroepithelial apical endfeet of the chick embryonic spinal cord, including sub-apical protrusions that extend laterally within the tissue, and observe similar structures in human neuroepithelium. We characterise the dynamics, shape, and cytoskeleton of these lateral protrusions and distinguish these structures from cytonemes/filopodia and tunnelling nanotubes. We demonstrate that lateral protrusions form a latticework of membrane contacts between non-adjacent cells, depend on actin but not microtubule dynamics and provide a lamellipodial-like platform for further extending fine actin-dependent filipodia. We find that lateral protrusions depend on the actin-binding protein WAVE1: mutant-WAVE1 misexpression attenuated prot...

Investigative Ophthalmology & Visual Science, 2003

Wellcome Open Research, 2019

Background:Atopic eczema is an itchy inflammatory disorder characterised by skin barrier dysfunct... more Background:Atopic eczema is an itchy inflammatory disorder characterised by skin barrier dysfunction. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a major risk factor, but the mechanisms by which filaggrin haploinsufficiency leads to atopic inflammation remain incompletely understood. Skin as an organ that can be modelled using primary cellsin vitroprovides the opportunity for selected genetic effects to be investigated in detail.Methods:Primary human keratinocytes and donor-matched primary fibroblasts from healthy individuals were used to create skin organoid models with and without siRNA-mediated knockdown ofFLG. Biological replicate sets of organoids were assessed using histological, functional and biochemical measurements.Results:FLGknockdown leads to subtle changes in histology and ultrastructure including a reduction in thickness of the stratum corneum and smaller, less numerous keratohyalin granules. Immature organoids showed some limited evidence of ba...

Mutations enhancing the kinase activity of LRRK2 cause Parkinson’s disease (PD) and therapies tha... more Mutations enhancing the kinase activity of LRRK2 cause Parkinson’s disease (PD) and therapies that reduce LRRK2 kinase activity are being tested in clinical trials. Numerous rare variants of unknown clinical significance have been reported, but how the vast majority impact on LRRK2 function is unknown. Here, we investigate 100 LRRK2 variants linked to PD, including previously described pathogenic mutations. We identify 23 LRRK2 variants that robustly stimulate kinase activity, including variants within the N-terminal non-catalytic regions [ARM (E334K, A419V), ANK(R767H), LRR (R1067Q, R1325Q)], as well as variants predicted to destabilise the ROC:CORB interface [ROC (A1442P, V1447M), CORA (R1628P) CORB (S1761R, L1795F)] and COR:COR dimer interface [CORB (R1728H/L)]. Most activating variants decrease LRRK2 biomarker site phosphorylation (pSer935/pSer955/pSer973), consistent with the notion that the active kinase conformation blocks their phosphorylation. We conclude that the impact of...

www.embojournal.org Structural insights into the regulation of PDK1 by phosphoinositides and inos... more www.embojournal.org Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates

After 2 h at 37°C, migrated or input cells were recovered from the wells, quantitated by flow cyt... more After 2 h at 37°C, migrated or input cells were recovered from the wells, quantitated by flow cytometry, and expressed as ratios of migrated or input HeN/HeJ. (B) DC were pretreated with 100 ng/ml LPS for different lengths of time before addition into the Transwell insert. Note that time = 0* data were obtained from cells where LPS was added immediately before the cells were placed onto the filter. Subsequent migration was for 2 h in all cases. The line indicates the 1:1 ratio achieved where HeN and HeJ migrate with equal efficiency. (C) Data from experiments on three or four independent DC cultures are shown. Bars represent mean data. *, P < 0.05.<b>Copyright information:</b>Taken from "TLR ligand–induced podosome disassembly in dendritic cells is ADAM17 dependent"The Journal of Cell Biology 2008;182(5):993-1005.Published online 8 Sep 2008PMCID:PMC2528573.

Investigative Ophthalmology & Visual Science, 2005