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Lrrn1 is required for formation of the midbrain-hindbrain boundary and organiser through regulation of affinity differences between midbrain and hindbrain cells in chick
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2011 (English)In: Developmental Biology, ISSN 0012-1606, E-ISSN 1095-564X, Vol. 352, no 2, p. 341-352Article in journal (Refereed) Published
Abstract [en]

The midbrain-hindbrain boundary (MHB) acts as an organiser/signalling centre to pattern tectal and cerebellar compartments. Cells in adjacent compartments must be distinct from each other for boundary formation to occur at the interface. Here we have identified the leucine-rich repeat (LRR) neuronal 1 (Lrrn1) protein as a key regulator of this process in chick. The Lrrn family is orthologous to the Drosophila tartan/capricious (trn/caps) family. Differential expression of trn/caps promotes an affinity difference and boundary formation between adjacent compartments in a number of contexts; for example, in the wing, leg and eye imaginal discs. Here we show that Lrrn1 is expressed in midbrain cells but not in anterior hindbrain cells. Lrrn1 is down-regulated in the anterior hindbrain by the organiser signalling molecule FGF8, thereby creating a differential affinity between these two compartments. Lrrn1 is required for the formation of MHB--loss of function leads to a loss of the morphological constriction and loss of Fgf8. Cells overexpressing Lrrn1 violate the boundary and result in a loss of cell restriction between midbrain and hindbrain compartments. Lrrn1 also regulates the glycosyltransferase Lunatic Fringe, a modulator of Notch signalling, maintaining its expression in midbrain cells which is instrumental in MHB boundary formation. Thus, Lrrn1 provides a link between cell affinity/compartment segregation, and cell signalling to specify boundary cell fate.

Place, publisher, year, edition, pages
Elsevier, 2011. Vol. 352, no 2, p. 341-352
Keyword [en]
Lrrn1, Tartan, Capricious, Boundary, Midbrain–hindbrain boundary organiser
National Category
Neurosciences Developmental Biology
Identifiers
URN: urn:nbn:se:umu:diva-57023DOI: 10.1016/j.ydbio.2011.02.002ISI: 000289180200015PubMedID: 21315708OAI: oai:DiVA.org:umu-57023DiVA, id: diva2:538849
Available from: 2012-07-02 Created: 2012-07-02 Last updated: 2018-05-04Bibliographically approved
In thesis
1. The release of histone proteins from cells via extracellular vesicles
Open this publication in new window or tab >>The release of histone proteins from cells via extracellular vesicles
2018 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Histones are chromatin-associated proteins localized to the nucleus. However, extracellular histones are present in biofluids from healthy individuals and become elevated under disease conditions, such as neurodegeneration and cancer. Hence, extracellular histones may have important biological functions in healthy and diseased states, which are not understood. Histones have been reported in the proteomes of extracellular vesicles (EVs), including microvesicles and exosomes. The main aim of this thesis was to determine whether or not extracellular histones are secreted via EVs/exosomes.

In an initial study (Paper I), I optimized methods for human embryonic kidney (HEK293) cell culture, transfection and protein detection using western blotting.

In the main study (Paper II), I used oligodendrocyte cell lines (rat OLN-93 and mouse Oli-neu) to investigate the localization of histones to EVs. Western blotting of EVs purified from OLN-93 cell-conditioned media confirmed the presence of linker and core histones in them. Immunolocalization and transmission electron microscopy confirmed that histones are localized to EVs, as well as intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). This suggests that histones are secreted via the MVB/exosome pathway.

Localization of histones in EVs was investigated by biochemical/proteolytic degradation and purification followed by western blotting. Surprisingly, histones were associated with the membrane but not the luminal fraction. Overexpression of tagged histones in HEK293 cells confirmed their conserved, membrane localization. OLN-93 cell EVs contained both double stranded and single stranded DNA but nuclease and protease digestion showed that the association of histones and DNA with EVs was not interdependent.

The abundance of histones in EVs was not affected by differentiation in Oli-neu cells. However, histone release was upregulated as an early response to cellular stress in OLN-93 cells and occurred before the release of markers of stress including heat shock proteins. Interestingly, a notable upregulation in secretion of small diameter (50-100 nm) EVs was observed following heat stress, suggesting that a sub-population of vesicles may be involved specifically in histone secretion in response to stress. Proteomic analyses identified the downregulation of endosomal sorting complex required for transport (ESCRT) as a possible mechanism underlying increased histone secretion.

In Paper III, I developed methods to quantify extracellular histone proteins in human ascites samples from ovarian cancer patients.

 

In summary, we show for the first time that membrane-associated histones are secreted via the MVB/exosome pathway. We demonstrate a novel pathway for extracellular histone release that may have a role in both health and disease. 

Place, publisher, year, edition, pages
Umeå: Umeå University, 2018. p. 74
Keyword
Histone, extracellular vesicles, exosomes, extracellular histones extracellular DNA, cellular stress, proteomics, ESCRT complex, Lrrn1, mid-hindbrain organizer, ovarian cancer, ascites
National Category
Cell and Molecular Biology Cell Biology
Identifiers
urn:nbn:se:umu:diva-147409 (URN)978-91-7601-888-0 (ISBN)
Presentation
2018-05-28, E04_ R-1, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2018-05-04 Created: 2018-05-03 Last updated: 2018-05-08Bibliographically approved

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