Peter Munro - Academia.edu (original) (raw)

Papers by Peter Munro

Chemical communications (Cambridge, England), Jan 26, 2017

Correlative NanoSIMS and EM imaging of amiodarone-treated macrophages shows the internalisation o... more Correlative NanoSIMS and EM imaging of amiodarone-treated macrophages shows the internalisation of the drug at a sub-cellular level and reveals its accumulation within the lysosomes, providing direct evidence for amiodarone-induced phospholipidosis. Chemical fixation using tannic acid effectively seals cellular membranes aiding intracellular retention of diffusible drugs.

Cell motility and the cytoskeleton, 2006

The classical Arp2/3-mediated dendritic network defines the cytoskeleton at the leading edge of c... more The classical Arp2/3-mediated dendritic network defines the cytoskeleton at the leading edge of crawling cells, and it is generally assumed that Arp2/3-mediated actin polymerization generates the force necessary to extend lamellipods. Our previous work suggested that successful lamellipod extension required not only free barbed ends for actin polymerization but also a proper ultrastructural organization of the cytoskeleton. To further explore the structural role of the Arp2/3 complex-mediated networks in lamellipod morphology and function, we performed a detailed analysis of the ultrastructure of the Arp2/3-mediated networks, using the WA domains of Scar and WASp to generate mislocalised Arp2/3 networks in vivo, and to reconstruct de novo Arp2/3-mediated actin nucleation and polymerization on extracted cytoskeletons. We present here evidence that spatially unrestricted Arp2/3-mediated networks are intrinsically three-dimensional and multilayered by nature and, as such, cannot sustai...

Cornea, 2005

To describe the clinical and ultrastructural features of 3 cases of acute corneal calcification f... more To describe the clinical and ultrastructural features of 3 cases of acute corneal calcification following accidental chemical injury. Three men presented over an 18-month period with unilateral eye injuries sustained when applying an industrial fire retardant. This product is predominantly a gypsum aggregate (calcium sulfate dihydrate) plaster combined under pressure with a set-time accelerator (aluminum sulfate). In each case the tear pH was initially alkaline, and the eyes were irrigated with phosphate-buffered saline according to protocol. Within hours a dense corneal opacity had developed that showed only minor resolution over 3 years of follow-up. Two eyes required corneal graft surgery for visual rehabilitation. Light and electron microscopy and energy dispersive analysis of x-rays (EDAX) was performed on excised tissue. Light and electron microscopy showed dense mineralization of the anterior stroma with discrete crystalline deposits in the deeper stroma. EDAX of the crystals showed high emission peaks for calcium and phosphorus. The insolubility, elemental composition, and ultrastructural appearance suggest that the opacity was caused by calcium phosphate deposition. The absence of phosphorus from the listed components of the fire retardant suggests that the use of phosphate-buffered irrigation fluid or the subsequent use of phosphate-buffered drops may have contributed to the deposition of this insoluble crystalline deposit.

Cells

Fenestrae are transcellular plasma membrane pores that mediate blood–tissue exchange in specialis... more Fenestrae are transcellular plasma membrane pores that mediate blood–tissue exchange in specialised vascular endothelia. The composition and biogenesis of the fenestra remain enigmatic. We isolated and characterised the protein composition of large patches of fenestrated plasma membrane, termed sieve plates. Loss-of-function experiments demonstrated that two components of the sieve plate, moesin and annexin II, were positive and negative regulators of fenestra formation, respectively. Biochemical analyses showed that moesin is involved in the formation of an actin–fodrin submembrane cytoskeleton that was essential for fenestra formation. The link between the fodrin cytoskeleton and the plasma membrane involved the fenestral pore protein PV-1 and Na,K-ATPase, which is a key regulator of signalling during fenestra formation both in vitro and in vivo. These findings provide a conceptual framework for fenestra biogenesis, linking the dynamic changes in plasma membrane remodelling to the...

Stem cell research & therapy, Jan 13, 2018

The use of human pluripotent stem cell-derived retinal cells for cell therapy strategies and dise... more The use of human pluripotent stem cell-derived retinal cells for cell therapy strategies and disease modelling relies on the ability to obtain healthy and organised retinal tissue in sufficient quantities. Generating such tissue is a lengthy process, often taking over 6 months of cell culture, and current approaches do not always generate large quantities of the major retinal cell types required. We adapted our previously described differentiation protocol to investigate the use of stirred-tank bioreactors. We used immunohistochemistry, flow cytometry and electron microscopy to characterise retinal organoids grown in standard and bioreactor culture conditions. Our analysis revealed that the use of bioreactors results in improved laminar stratification as well as an increase in the yield of photoreceptor cells bearing cilia and nascent outer-segment-like structures. Bioreactors represent a promising platform for scaling up the manufacture of retinal cells for use in disease modelling...

Discoveries (Craiova, Romania)

Three subsets of mitochondria have been described in adult cardiomyocytes - intermyofibrillar (IM... more Three subsets of mitochondria have been described in adult cardiomyocytes - intermyofibrillar (IMF), subsarcolemmal (SSM), and perinuclear (PN). They have been shown to differ in physiology, but whether they also vary in morphological characteristics is unknown. Ischemic preconditioning (IPC) is known to prevent mitochondrial dysfunction induced by acute myocardial ischemia/reperfusion injury (IRI), but whether IPC can also modulate mitochondrial morphology is not known. Morphological characteristics of three different subsets of adult cardiac mitochondria along with the effect of ischemia and IPC on mitochondrial morphology will be investigated. Mouse hearts were subjected to the following treatments (N=6 for each group): stabilization only, IPC (3x5 min cycles of global ischemia and reperfusion), ischemia only (20 min global ischemia); and IPC and ischemia. Hearts were then processed for electron microscopy and mitochondrial morphology was assessed subsequently. In adult cardiomyo...

Human molecular genetics, Jul 5, 2017

Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy. We recently ide... more Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy. We recently identified mutations in REEP6, which encodes the receptor expression enhancing protein 6, in several families with autosomal recessive RP. REEP6 is related to the REEP and Yop1p family of ER shaping proteins and potential receptor accessory proteins, but the role of REEP6 in the retina is unknown. Here we characterise the disease mechanisms associated with loss of REEP6 function using a Reep6 knockout mouse generated by CRISPR/Cas9 gene editing. In control mice REEP6 was localised to the inner segment and outer plexiform layer of rod photoreceptors. The Reep6-/- mice exhibited progressive photoreceptor degeneration from P20 onwards. Ultrastructural analyses at P20 by transmission electron microscopy and 3View serial block face scanning EM revealed an expansion of the distal ER in the Reep6-/- rods and an increase in their number of mitochondria. Electroretinograms revealed photoreceptor dys...

Human molecular genetics, Jan 7, 2017

Protein misfolding caused by inherited mutations leads to loss of protein function and potentiall... more Protein misfolding caused by inherited mutations leads to loss of protein function and potentially toxic 'gain of function', such as the dominant P23H rhodopsin mutation that causes retinitis pigmentosa (RP). Here, we tested whether the AMPK activator metformin could affect the P23H rhodopsin synthesis and folding. In cell models, metformin treatment improved P23H rhodopsin folding and traffic. In animal models of P23H RP, metformin treatment successfully enhanced P23H traffic to the rod outer segment, but this led to reduced photoreceptor function and increased photoreceptor cell death. The metformin-rescued P23H rhodopsin was still intrinsically unstable and led to increased structural instability of the rod outer segments. These data suggest that improving the traffic of misfolding rhodopsin mutants is unlikely to be a practical therapy, because of their intrinsic instability and long half-life in the outer segment, but also highlights the potential of altering translatio...

Archives of Ophthalmology, Jul 1, 1998

To investigate the ultrastructural features of idiopathic full-thickness macular hole (FTMH) oper... more To investigate the ultrastructural features of idiopathic full-thickness macular hole (FTMH) opercula excised during vitrectomy and to correlate them with the outcome of surgery. Opercula were collected from eyes undergoing vitrectomy for stage 3 FTMH using noncrushing, cupped foreign body forceps. Following immediate fixation, specimens were processed for transmission electron microscopy. The ultrastructural features were correlated with the clinical data recorded for each patient before and after surgery. Eighteen specimens were studied. Native vitreous collagen was identified on the surface of all 18, while fragments of internal limiting membrane were present in 11 (61%). Eleven (61%) were found to contain only glia, comprising fibrous astrocytes and Müller cells in variable proportions. The remaining 7 (39%) were found to contain, in addition to glia, neurites and synaptic complexes, of which some were typical of cone photoreceptors. The initial surgical closure rate was significantly better in eyes in which only glia were present (9/11 [82%]), compared with those with neurites (1/7 [14%]) (P = .01). Once closure had been achieved with reoperation, the median final visual acuity was 20/60 in both groups (P = .26), although the likelihood of achieving an acuity of 20/40 or better was greater in the former (50%) than the latter group (17%). Two distinct types of opercula occur in association with stage 3 FTMH--those containing only glia (pseudo-opercula), which are probably associated with a foveal dehiscence and little or no loss of foveal tissue, and those containing both glia and a significant number of avulsed foveal cones (true opercula), which arise from a full-thickness foveal tear. Although the loss of foveal tissue in true opercula would seem to explain the worse initial anatomical and more modest visual results in some eyes, significant visual improvement may still be achieved after successful closure. The presence of neurites in true opercula suggests that, in at least some cases, direct traction on the foveal retina leads to macular hole formation.

Investigative Ophthalmology Visual Science, Mar 1, 2002

PURPOSE. To characterize a spontaneously immortalized human Müller cell line and to determine whe... more PURPOSE. To characterize a spontaneously immortalized human Müller cell line and to determine whether it retains the characteristics of primary isolated cells without undergoing differentiation in vitro. METHODS. An immortalized cell line obtained from human retina was investigated for the expression of known markers of Müller cells, including cellular retinaldehyde binding protein (CRALBP), glutamine synthetase, epidermal growth factor receptor (EGF-R), ␣-smooth muscle actin (␣-SMA), and glial fibrillary acidic protein (GFAP). Also examined were the morphologic features of these cells, by scanning and transmission electron microscopy, and their functional characteristics, by electrogenic responses to glutamate. In addition, comparative studies were made of these cells with primary cultures of freshly isolated human Müller cells. RESULTS. The cells expressed CRALBP, EGF-R, glutamine synthetase, and ␣-SMA, as judged by confocal microscopy and Western blot analysis of cell lysates. Western blot analysis did not detect GFAP in cell lysates, but confocal microscopy showed that occasional cells expressed GFAP after detachment from the monolayer. The morphologic features of the cells examined, as judged by scanning and transmission electron microscopy, resemble those of cells derived from primary cell cultures. They possess villous projections on their apical surfaces and contain loose bundles of microtubules aligned parallel to one another and the long axis of the cell process. Characteristically, they contain abundant deposits of glycogen particles that do not differ from those seen in primary isolated cells. Preliminary recordings with intracellular electrodes revealed that these cells have properties similar to those described for mammalian Müller cells and depolarize in response to L-glutamate without significant change in membrane resistance, consistent with the well-established electrogenic uptake of this amino acid. CONCLUSIONS. A spontaneously immortalized Müller cell line was characterized that retains the characteristics of primary isolated cells in culture. To the authors' knowledge, it constitutes the first human Müller cell line reported in the literature. It has been named MIO-M1 (Moorfields/Institute of Ophthalmology-Müller 1) after the authors' institution. Availability of this human cell line will facilitate studies designed to obtain a better understanding of the role of Müller cells in normal and pathologic conditions.

Nature Genetics 37 Pp 1135 1140, Oct 1, 2005

Curr Eye Res 17 917 923, Sep 1, 1998

This study was aimed at determining whether or not apoptotic photoreceptor cell death in a mouse ... more This study was aimed at determining whether or not apoptotic photoreceptor cell death in a mouse model of inherited retinal degeneration is p53 dependent. A colony of p53-deficient rds mice were obtained by crossing homozygous rds mice with animals homozygous for a targeted disruption of the p53 gene and genotyping the offspring of the F1 cross. Both parental strains were on a BALB/c background. Age matched p53-deficient rds mice and controls (p53-deficient, rds and BALB/c mice), were sacrificed from day 1 to day 58 after birth. Eyes were paraffin-embedded and a modified terminal dUTP nick-end labeling (TUNEL) technique was used to detect the number of cells displaying DNA fragmentation within the sectioned retina. Eyes were also resin-embedded for semi-thin and ultra-thin sectioning. The peak in photoreceptor apoptosis, which occurs at 16 days in the rds mouse, was delayed by 3 days in p53-deficient rds mice. In addition, there was also a delay in the loss of photoreceptor cells between 16 and 26 days. However, absence of p53 did not prevent retinal degeneration in the rds mouse. The number of photoreceptor cells in p53-deficient rds mice at 35 days was very similar to that in the controls. We have demonstrated that absence of p53 delays but does not prevent photoreceptor cell loss in the rds mouse. Our results provide evidence for plasticity in the mechanism by which apoptosis proceeds in retinal degeneration.

eLife, Jan 26, 2016

Epithelial fusion is a crucial process in embryonic development, and its failure underlies severa... more Epithelial fusion is a crucial process in embryonic development, and its failure underlies several clinically important birth defects. For example, failure of neural fold fusion during neurulation leads to open neural tube defects including spina bifida. Using mouse embryos, we show that cell protrusions emanating from the apposed neural fold tips, at the interface between the neuroepithelium and the surface ectoderm, are required for completion of neural tube closure. By genetically ablating the cytoskeletal regulators Rac1 or Cdc42 in the dorsal neuroepithelium, or in the surface ectoderm, we show that these protrusions originate from surface ectodermal cells and that Rac1 is necessary for the formation of membrane ruffles which typify late closure stages, whereas Cdc42 is required for the predominance of filopodia in early neurulation. This study provides evidence for the essential role and molecular regulation of membrane protrusions prior to fusion of a key organ primordium in ...

Cell Stem Cell, 2016

If citing, it is advised that you check and use the publisher's definitive version for pagination... more If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections.

Journal of leukocyte biology, Jan 20, 2015

The immunological synapse is a highly structured and molecularly dynamic interface between commun... more The immunological synapse is a highly structured and molecularly dynamic interface between communicating immune cells. Although the immunological synapse promotes T cell activation by dendritic cells, the specific organization of the immunological synapse on the dendritic cell side in response to T cell engagement is largely unknown. In this study, confocal and electron microscopy techniques were used to investigate the role of dendritic cell actin regulation in immunological synapse formation, stabilization, and function. In the dendritic cell-restricted absence of the Wiskott-Aldrich syndrome protein, an important regulator of the actin cytoskeleton in hematopoietic cells, the immunological synapse contact with T cells occupied a significantly reduced surface area. At a molecular level, the actin network localized to the immunological synapse exhibited reduced stability, in particular, of the angiopoietin-related protein-2/3-dependent, short-filament network. This was associated w...

Investigative ophthalmology & visual science, 2002

To characterize a spontaneously immortalized human Müller cell line and to determine whether it r... more To characterize a spontaneously immortalized human Müller cell line and to determine whether it retains the characteristics of primary isolated cells without undergoing differentiation in vitro. An immortalized cell line obtained from human retina was investigated for the expression of known markers of Müller cells, including cellular retinaldehyde binding protein (CRALBP), glutamine synthetase, epidermal growth factor receptor (EGF-R), alpha-smooth muscle actin (alpha-SMA), and glial fibrillary acidic protein (GFAP). Also examined were the morphologic features of these cells, by scanning and transmission electron microscopy, and their functional characteristics, by electrogenic responses to glutamate. In addition, comparative studies were made of these cells with primary cultures of freshly isolated human Müller cells. The cells expressed CRALBP, EGF-R, glutamine synthetase, and alpha-SMA, as judged by confocal microscopy and Western blot analysis of cell lysates. Western blot anal...

Journal of cell science, Jan 15, 2002

Mutations in the photopigment rhodopsin are the major cause of autosomal dominant retinitis pigme... more Mutations in the photopigment rhodopsin are the major cause of autosomal dominant retinitis pigmentosa. The majority of mutations in rhodopsin lead to misfolding of the protein. Through the detailed examination of P23H and K296E mutant opsin processing in COS-7 cells, we have shown that the mutant protein does not accumulate in the Golgi, as previously thought, instead it forms aggregates that have many of the characteristic features of an aggresome. The aggregates form close to the centrosome and lead to the dispersal of the Golgi apparatus. Furthermore, these aggregates are ubiquitinated, recruit cellular chaperones and disrupt the intermediate filament network. Mutant opsin expression can disrupt the processing of normal opsin, as co-transfection revealed that the wild-type protein is recruited to mutant opsin aggregates. The degradation of mutant opsin is dependent on the proteasome machinery. Unlike the situation with DeltaF508-CFTR, proteasome inhibition does not lead to a mar...

Cell Differentiation and Development, 1989

We have found that glial progenitor cells isolated from the optic nerves of adult rats are fundam... more We have found that glial progenitor cells isolated from the optic nerves of adult rats are fundamentally different from their counterparts in perinatal animals. In our studies on bipotential oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, we have seen that O-2A ad "" progenitor cells can be distinguished from O-2S. peril "" a ' progenitors by their morphology and antigenic phenotype, their much longer cell cycle time (65 h versus 18 h), slower rate of migration rate (4fimh-1 versus 21/im h" 1), and their time course of differentiation into oligodendrocytes or type-2 astrocytes in vitro (=S3 days versus >5 days). At least some of the differences between O-2A a</u " and O-2A perir "" aI progenitor cells appear to be clearly related to the differing cellular requirements of the adult and perinatal central nervous system (CNS). The properties of the O-2\ adult progenitor cells may make these cells ideally suited for the needs of the adult CNS, where rapid exponential increases in the number of oligodendrocytes and O-2A progenitor cells would be inappropriate. However, the properties of the O-2A a ' / "" progenitor cells are such that they may not be able to replace oligodendrocytes in sufficient numbers to repair extensive or recurrent damage in the adult brain, such as in patients suffering from the human demyelinating disease multiple sclerosis. Moreover, available information about other tissues suggests that the transition from perinatal to adult progenitor cell types may represent a developmental mechanism of general importance.

Human molecular genetics, 2015

Understanding phenotype-genotype correlations in retinal degeneration is a major challenge. Mutat... more Understanding phenotype-genotype correlations in retinal degeneration is a major challenge. Mutations in CRB1 lead to a spectrum of autosomal recessive retinal dystrophies with variable phenotypes suggesting the influence of modifying factors. To establish the contribution of the genetic background to phenotypic variability associated with the Crb1(rd8/rd8) mutation, we compared the retinal pathology of Crb1(rd8/rd8)/J inbred mice with that of two Crb1(rd8/rd8) lines backcrossed with C57BL/6JOlaHsd mice. Topical endoscopic fundal imaging and scanning laser ophthalmoscopy fundus images of all three Crb1(rd8/rd8) lines showed a significant increase in the number of inferior retinal lesions that was strikingly variable between the lines. Optical coherence tomography, semithin, ultrastructural morphology and assessment of inflammatory and vascular marker by immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction revealed that the lesions were associated wit...