Trevor Sherwin

Assembly of the paraflagellar rod and the flagellum attachment zone complex during the Trypanosoma brucei cell cycle.

ABSTRACT Trypanosomes possess a single flagellum that is attached to their cell body via the flagellum attachment zone (FAZ). The FAZ is composed of two structures: a cytoplasmic filament complex and four microtubules situated next to it. There is a complex transmembrane crosslinking of this FAZ to the paraflagellar rod (PFR) and axoneme within the flagellum. We have partially purified the FAZ complex and have produced monoclonal antibodies both against the FAZ and the paraflagellar rod. The two antibodies against the FAZ (L3B2 and L6B3) recognise the cytoplasmic filament in immunofluorescence and in immunoelectron microscopy. On Western blot, they detect a doublet of high molecular weight (M, 200,000). Two anti‐PFR antibodies (L13D6 and L8C4) recognise the paraflagellar rod in immunofluorescence, but show a difference on Western blot: L13D6 recognises both major PFR proteins, whereas L8C4 is specific for only one of them. Using these new antibodies we have shown that although the growth of both cytoplasmic FAZ filament and external PFR are related, their growth initiates at different time points during the cell cycle and the two structures elongate at distinct rates.

Paraflagellar rod is vital for trypanosome motility

African trypanosomes are protozoan parasites that cause sleeping sickness in man. In addition to the axoneme, their flagellum contains a large structure called the paraflagellar rod (PFR) whose function is unknown. We used an antisense RNA approach to produce a specific molecular ablation of the PFR structure. The mutant cells are paralysed, demonstrating that the PFR has an essential role in cell motility.

Visualization of detyrosination along single microtubules reveals novel mechanisms of assembly during cytoskeletal duplication in trypanosomes

We have been able to use immunogold labeling with monoclonal antibodies specific for tyrosinated alpha-tubulin to define new microtubule assembly within the T. brucei pellicular cytoskeleton. Using this approach, we have been able to visualize and define the detyrosination gradient along single microtubules in vivo. New microtubules are seen to invade the cytoskeletal array early in the cell cycle between old microtubules. In post-mitotic cells, a unique form of microtubule assembly occurs, with very short microtubules being intercalated in the array. We propose that these are nucleated by lateral interaction with the MAPs on existing adjacent microtubules. This construction pattern suggests a templated morphogenesis of microtubule arrays with semi-conservative distribution to the daughter cells.

Age-related differences in the normal human cornea: a laser scanning in vivo confocal microscopy study

Aims: To quantify and establish baseline normative data for age-related differences in cellular and innervation density in the normal, healthy, human cornea using laser scanning in vivo confocal microscopy. Methods: Cross-sectional study of 85 normal subjects assessed via corneal topography and laser scanning in vivo confocal microscopy. Results: Mean age was 38±16 years (range 18–87 years) and 60% of subjects were female. Anterior keratocyte density declined by 0.9% per year (r = −0.423, p<0.001), posterior keratocyte density declined by 0.3% per year (r = −0.250, p = 0.021) and endothelial cell density declined by 0.5% per year (r = −0.615, p<0.001). Sub-basal nerve fibre density declined by 0.9% per year (r = −0.423, p<0.001). No association was observed between age and basal epithelial cell density, or between age and central corneal thickness, corneal astigmatism or horizontal corneal diameter (p>0.05). No association was observed between subject gender and corneal cell or innervation density. Conclusions: Using laser scanning in vivo confocal microscopy this study highlights a significant, and relatively linear, reduction in keratocyte and endothelial cell density with increasing subject age. Interestingly, corneal sub-basal nerve fibre density also significantly decreases with increasing age. In vivo laser scanning confocal microscopy provides a safe, non-invasive method for the establishment of normative data and assessment of alterations in human corneal microstructure following surgery or disease processes.

Architecture of the Trypanosoma brucei nucleus during interphase and mitosis

Abstract.The structural basis of mitosis, spindle organisation and chromosome segregation, in the unicellular parasite Trypanosoma brucei is poorly understood. Here, using immunocytochemistry, fluorescent in situ hybridisation and electron microscopy, we provide a detailed analysis of mitosis in this parasite. We describe the organisation of the mitotic spindle during different stages of mitosis, the complex ultrastructure of kinetochores and the identification of a potential spindle-organising centre in the mitotic nucleus. We investigate the dynamics of chromosome segregation using telomeric and chromosome-specific probes. We also discuss the problems involved in chromosome segregation in the light of the fact that the T. brucei karyotype has 22 chromosomes in the apparent presence of only eight ultrastructurally defined kinetochores.

Laser scanning in vivo confocal microscopy reveals reduced innervation and reduction in cell density in all layers of the keratoconic cornea.

PURPOSE The exact pathophysiological processes underlying keratoconus remain an enigma. In this study, laser scanning in vivo confocal microscopy (IVCM) was used to define further the microstructural abnormalities in the keratoconic cornea and to establish the relationship with disease severity. METHODS This was a prospective, cross-sectional study comparing 52 subjects with keratoconus and 52 age-matched control subjects. Assessment included demographics, history, slit lamp biomicroscopy, computerized corneal tomography, and laser scanning IVCM. RESULTS Significantly lower cell densities (in cells per square millimeter, mean +/- SD) were observed in keratoconus corneas than in normal ones: basal epithelial cells, 4340.6 +/- 595.2 vs. 5777.6 +/- 958.2 (P < 0.001), anterior keratocytes, 523.6 +/- 206.4 vs. 859.7 +/- 219.1 (P < 0.001), posterior keratocytes, 240.4 +/- 64.5 vs. 330.6 +/- 52.3 (P < 0.001), and endothelial cells 2412.2 +/- 339.5 vs. 2845.6 +/- 313.0 (P < 0.001). Subbasal nerve fiber density was 52.7% lower in keratoconus corneas than in the control (P < 0.001). Basal epithelial cell density (P = 0.001), subbasal nerve fiber density (P = 0.015), and anterior keratocyte density (P < 0.001) correlated with severity of disease. Lower subbasal nerve density also correlated with younger age at diagnosis (r = 0.397, P = 0.004). Severe disease was associated with diagnosis at a younger age (P = 0.023), a history of eye rubbing (P = 0.025), and Maori or Pacific Island ethnicity (P = 0.001). CONCLUSIONS Significant microstructural abnormalities were identified at every level of the keratoconic cornea and were related to disease severity. IVCM offers a potential insight into the pathophysiology of the microstructural changes in keratoconus.

A motility function for the paraflagellar rod of Leishmania parasites revealed by PFR-2 gene knockouts.

We demonstrate a functional role for the paraflagellar rod (PFR) in motility of Leishmania mexicana. The PFR is a complex cytoskeletal structure running parallel to the axoneme in the flagella of kinetoplastid protozoa. The PFR is composed of a latticework of protein filaments whose major constituents are two related proteins (PFR-1 and PFR-2 in Leishmania). The molecular details of their assembly into PFR filaments are unknown as is the biological function of the PFR. As an approach to understanding the structure and function of the PFR in Leishmania, we made L. mexicana null mutants of PFR-2. PFR-2 minus parasites grow and divide normally in culture and still express the PFR-1 protein. They lack most of the PFR structure demonstrating that the PFR-2 protein is an essential constituent of the PFR. Detailed ultrastructural analysis of the PFR-2 null mutant reveals the presence of a residual inner substructure of the PFR which contains PFR-1 protein, indicating that PFR-1 can polymerize in the absence of PFR-2. The PFR-2 null mutant displays pronounced changes in flagellar beat waveform and forward swimming velocity, compared to wild type parasites consistent with decreased internal elastic bending resistance in PFR-lacking flagella, and indicating a functional role for the PFR in the motility of Leishmania.

Morphological changes in keratoconus: pathology or pathogenesis

Keratoconus was first discriminated from other corneal ectatic diseases in 1854. Since that time the morphological characteristics of keratoconic progression have been invaluable in the diagnosis of the condition. The key clinical features used to identify keratoconus have remained essentially the same since the introduction of the slit‐lamp biomicroscope. Only relatively recently has the development of computerized corneal topography revolutionized the diagnosis of early keratoconus. Analysis of peer‐reviewed literature databases revealed a steady chronological increase in pathological research into the progress of keratoconus. This overview describes the recent advances in our understanding of keratoconic pathology and highlights the interactions within the cornea that may be important in the pathogenesis of this condition.

Microtubular organization visualized by immunofluorescence microscopy during erythrocytic schizogony in Plasmodium falciparum and investigation of post-translational modifications of parasite tubulin.

We describe a novel procedure for the immunofluorescent investigation of Plasmodium falciparum. This has allowed us to visualize clearly microtubular structures and their changing conformation through the erythrocytic cell-cycle, to the stage of cytodifferentiation leading to merozoite release. The images of spindle development we observed, together with an analysis of nuclear body numbers in large numbers of parasites, indicate that there is an apparent asynchrony in chromosomal multiplication within a single parasite. Using antibodies specific for post-translational modification of alpha-tubulin, we also demonstrate that the C-terminal tyrosine-containing epitope of P. falciparum alpha-tubulin I is similar to that of other organisms. Lysine-40 in the same molecule, a target for highly specific in vivo acetylation in some organisms, is unmodified in the blood stages we examined here. After in vitro acetylation of this residue, however, the epitope to which it contributes was recognized by antibody, showing that the conformation of this part of the molecule is also conserved, despite a lack of primary sequence homology immediately downstream of the target lysine residue.

Acute Wound Healing in the Human Central Corneal Epithelium Appears to Be Independent of Limbal Stem Cell Influence

PURPOSE In the adult cornea, epithelial cells are maintained by limbal stem cells (LSCs) that cycle slowly and give rise to transient amplifying (TA) cells. These migrate centripetally, differentiate outward to the surface, and are then lost by desquamation. This study was conducted to analyze the contribution of human central corneal epithelial cells toward corneal epithelial regeneration. METHODS A human corneal organotypic culture model was used to assess corneal healing in vitro in 12 matched cornea pairs. Two types of ablation were studied: (1) A ring-shaped, transepithelial, excimer laser (193 nm) ablation, of 7 mm outer diameter and 3 mm inner diameter, to a depth of 80 mum-sparing the central and peripheral corneal epithelium; and (2) an ablation pattern identical to that in (1) with ablation of the limbal epithelium in addition. Corneal healing was followed using time-lapse dark-field microscopy for up to 12 hours, and the corneas were analyzed by using immunohistochemical markers for cell proliferation and stem cells. RESULTS In the donut model, corneal epithelial repair originated from both the limbus and the central corneal epithelium with the average rate of epithelial recovery from the center being similar to the rate from the periphery (0.06 +/- 0.01 mm/h vs. 0.07 +/- 0.03 mm/h, P = 0.44). When the area of recovery was calculated relative to original edge circumferences, the central epithelial rate tended to be faster than the peripheral (0.06 +/- 0.02 mm(2)/mm/h vs. 0.04 +/- 0.01 mm(2)/mm/h, P = 0.04). Similar rates in epithelial recovery were identified in centripetal and centrifugal directions in both the donut and donut+limbus ablation models. Central epithelial cell density increased 36% over the control cornea within 12 hours after surgery, but there was no change at the periphery. Cell proliferation, assessed using Ki67 and BrdU labeling, was observed across the entire cornea. Expression of the putative stem cell markers p63 and ABCG2 was clearly evident in the basal layer of the limbus. However, weaker labeling was also observed in the central epithelium. Connexin 43 (Cx43), a differentiation marker, was mainly absent in the normal untreated limbal basal cells, but more Cx43-positive cells were labeled in the basal layer of the limbus after wounding. CONCLUSIONS After wounding, the capacity for epithelial cell proliferative and migration appears to be as active in the central cornea as in the periphery/limbus. Central and peripheral epithelial recovery remains equal even after ablation of the limbus. Central human corneal epithelial cells are therefore capable of corneal epithelial regeneration, at least in the first 12 hours after wounding.