TY  - JOUR
A1  - Lando, David
A1  - Endesfelder, Ulrike
A1  - Berger, Harald
A1  - Subramanian, Lakxmi
A1  - Dunne, Paul D.
A1  - McColl, James
A1  - Klenerman, David
A1  - Carr, Antony M.
A1  - Sauer, Markus
A1  - Allshire, Robin C.
A1  - Heilemann, Mike
A1  - Laue, Ernest D.
T1  - Quantitative single-molecule microscopy reveals that CENP-A\(^{Cnp1}\) deposition occurs during G2 in fission yeast
JF  - Open Biology
N2  - The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and 'counts' individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-A\(^{Cnp1}\) with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-A\(^{Cnp1}\) levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-A(Cnp1) is deposited solely during the G2 phase of the cell cycle.
KW  - nucleosome
KW  - fission yeast
KW  - identification
KW  - propagation
KW  - CSE4, CENP-A
KW  - CENP-A
KW  - schizosaccaromyces-pombe
KW  - fluorescent protein
KW  - centomeres
KW  - superresolution
KW  - chromatin
KW  - centromere
KW  - ingle-molecule microscopy
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134682
VL  - 2
IS  - 120078
ER  - 
TY  - JOUR
A1  - Giles, James A.
A1  - Greenhalgh, Andrew D.
A1  - Denes, Adam
A1  - Nieswandt, Bernhard
A1  - Coutts, Graham
A1  - McColl, Barry W.
A1  - Allan, Stuart M.
T1  - Neutrophil infiltration to the brain is platelet-dependent, and is reversed by blockade of platelet GPIbα
JF  - Immunology
N2  - Neutrophils are key components of the innate immune response, providing host defence against infection and being recruited to non-microbial injury sites. Platelets act as a trigger for neutrophil extravasation to inflammatory sites but mechanisms and tissue-specific aspects of these interactions are currently unclear. Here, we use bacterial endotoxin in mice to trigger an innate inflammatory response in different tissues and measure neutrophil invasion with or without platelet reduction. We show that platelets are essential for neutrophil infiltration to the brain, peritoneum and skin. Neutrophil numbers do not rise above basal levels in the peritoneum and skin and are decreased (~60%) in the brain when platelet numbers are reduced. In contrast neutrophil infiltration in the lung is unaffected by platelet reduction, up-regulation of CXCL-1 (2·4-fold) and CCL5 (1·4-fold) acting as a compensatory mechanism in platelet-reduced mice during lung inflammation. In brain inflammation targeting platelet receptor GPIbα results in a significant decrease (44%) in platelet-mediated neutrophil invasion, while maintaining platelet numbers in the circulation. These results suggest that therapeutic blockade of platelet GPIbα could limit the harmful effects of excessive inflammation while minimizing haemorrhagic complications of platelet reduction in the brain. The data also demonstrate the ability to target damaging brain inflammation in stroke and related disorders without compromising lung immunity and hence risk of pneumonia, a major complication post stroke. In summary, our data reveal an important role for platelets in neutrophil infiltration to various tissues, including the brain, and so implicate platelets as a key, targetable component of cerebrovascular inflammatory disease or injury.
KW  - brain
KW  - inflammation
KW  - neuroinflammation
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-233048
VL  - 154
ER  -