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P Holroyd

Publications and source records attributed to P Holroyd.

6 recordsLinked to original sources
PubMed2001

SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis.

During exocytosis, SNARE proteins of secretory vesicles interact with the corresponding SNARE proteins in the plasmalemma to initiate the fusion reaction. However, it is unknown whether SNAREs are uniformly distributed in the membrane or whether specialized fusion sites exist. Here we report that in the plasmalemma, syntaxins are concentrated in 200 nm large, cholesterol-dependent clusters at which secretory vesicles preferentially dock and fuse. The syntaxin clusters are distinct from cholesterol-dependent membrane rafts since they are Triton X-100-soluble and do not co-patch with raft markers. Synaptosomal-associated protein (SNAP)-25 is also clustered in spots, which partially overlap with syntaxin. Cholesterol depletion causes dispersion of these clusters, which is associated with a strong reduction in the rate of secretion, whereas the characteristics of individual exocytic events are unchanged. This suggests that high local concentrations of SNAREs are required for efficient fusion.

Animals↗
PubMed2000

A cell-free system for regulated exocytosis in PC12 cells.

We have developed a cell-free system for regulated exocytosis in the PC12 neuroendocrine cell line. Secretory vesicles were preloaded with acridine orange in intact cells, and the cells were sonicated to produce flat, carrier-supported plasma membrane patches with attached vesicles. Exocytosis resulted in the release of acridine orange which was visible as a disappearance of labeled vesicles and, under optimal conditions, produced light flashes by fluorescence dequenching. Exocytosis in vitro requires cytosol and Ca(2+) at concentrations in the micromolar range, and is sensitive to Tetanus toxin. Imaging of membrane patches at diffraction- limited resolution revealed that 42% of docked granules were released in a Ca(2+)-dependent manner during 1 min of stimulation. Electron microscopy of membrane patches confirmed the presence of dense-core vesicles. Imaging of membrane patches by atomic force microscopy revealed the presence of numerous particles attached to the membrane patches which decreased in number upon stimulation. Thus, exocytotic membrane fusion of single vesicles can be monitored with high temporal and spatial resolution, while providing access to the site of exocytosis for biochemical and molecular tools.

Acridine Orange↗
PubMed2000

Live cell imaging by multifocal multiphoton microscopy.

Multifocal multiphoton microscopy (MMM) permits parallel multiphoton excitation by scanning an array of high numerical aperture foci across a plane in the sample. MMM is particularly suitable for live cell investigations since it combines advantages of standard multiphoton microscopy such as optical sectioning and suppression of out-of-focus phototoxicity with high recording speeds. Here we describe several applications of MMM to live cell imaging using the neuroendocrine cell line PC12 and bovine chromaffin cells. Stainings were performed with the acidophilic dye acridine orange and the lipophilic dyes FM1-43 and Fast DiA as well as by transfection of the cells with GFP. In both bovine chromaffin and PC12 cells structural elements of nuclear chromatin and the 3-D distribution of acidic organelles inside the cells were visualized. In PC12 cells differentiated by nerve growth factor examples of neurites were monitored. Stainings of membranes were used to reconstruct the morphology of cells and neurites in three dimensions by volume-rendering and by isosurface plots. 3-D reconstructions were composed from stacks of about 50 images each with a diameter of 30-100 microm that were acquired within a few seconds. We conclude that MMM proves to be a technically simple and very effective method for fast 3-D live cell imaging at high resolution.

Acridine Orange↗
PubMed1979

A method for the study of therapeutic factors in group psychotherapy.

A method is described for the study of therapeutic factors in group therapy in which patients and their therapists prepare brief reports at regular intervals about those events in treatment which they regard as important; these reports are then assigned by independent judges to a classification of therapeutic factors which has been specifically devised for this purpose. The feasibility, validity and reliability of the method are discussed and its potential application to group therapy research and training briefly mentioned.

Adult↗
PubMed1976

The Isis Centre: a counselling service within the National Health Service.

A new centre has been established to provide readily accessible counselling, consultation, and mental health information. People may refer themselves or are recommended to attend by general practitioners or other agencies. The counsellors have varied backgrounds in paramedical or counselling services, and they are supported by psychiatrists. Of a sample of 100 clients, four were referred to one of the team's psychiatrists and 33 visited the centre only once. The centre's staff aim to adopt a flexible approach to the client and his problems, and formal psychiatric categories have not been found useful. Provision is made for people who want to solve their problems by discussion rather than medication and those for whom the existing psychiatric services may have little time to spare. Consequently, the approach adopted by the Isis Centre, whereby many people benefit from psychotherapy yet the psychiatrist deals directly with only a few selected cases, contributes towards meeting the great need for psychiatric services and using the psychiatrist's skills more effectively.

Adult↗