Visualizing lipid regulation of gene expression using new and classical imaging tools

0473398 - ÚMG 2017 RIV TH eng C - Conference Paper (international conference)
Hozák, Pavel
Visualizing lipid regulation of gene expression using new and classical imaging tools.
APMC11 Program and Abstract Book. Bangkok: Anatomy Association of Thailand, 2016. ISBN 978-616-279-846-7.
[11th Asia-pacific Microscopy Conference (APMC11). Phuket (TH), 23.05.2016-27.05.2016]
R&D Projects: GA ČR GA15-08738S; GA TA ČR(CZ) TE01020118; GA MŠMT(CZ) ED1.1.00/02.0109; GA ČR(CZ) GA16-03346S
Institutional support: RVO:68378050
Keywords : nucleus * PIP2 * chromatin * Pol II transcription
Subject RIV: EB - Genetics ; Molecular Biology

While fluorescent microscopy allows for simultaneous detection of multiple antigens, the electron microscopy (EM) sensitive immunodetection is limited to only two antigens. I will summarize the current possibilities of single molecule visualization inside of cells and tis-sues, and discuss future needs of researches in biomedicine. In order to overcome the current limitations of immunodetection, we prepared a set of novel nanoparticles (NPs) which fulfil several criteria: size in the frame of 5-12 nm, small size distribution, good contrast and stability in the electron microscope, stability of colloidal solution during conjugation, and surface properties allowing for conjugation with antibodies With the use of novel NPs, various combinations with commercial gold NPs can be made to obtain a set for simultaneous labelling. For the first time in ultrastructural histochemistry, up to five molecular targets can be identified simultaneously. Using our previously developed tools of spatial statistics one could then map the regions of distribution of multiple molecular targets within the cell, as well as to analyse a high number of individual molecular interactions. Also, we characterized some of the critical steps during (cryo)sample preparation in order to achieve the best preservation of both ultrastructure and antigen in cells.
These methods allowed us to progress with understanding some novel molecular interactions in the cell nucleus. The nucleus is a highly organized cell compartment, where controlled gene expression, DNA replication, and RNA processing occur. These processes are spatially ordered via the nucleoskeleton, which is involved in nuclear compartmentalization and critical for nuclear functioning. In spite of the growing interest and extensive research concerned to the nuclear organization, so far mostly protein complexes have been found as important for spatial nuclear ordering. We describe here novel structures containing phosphatidylinositol 4,5-bisphosphate (PIP2) which contribute as well. Based on scarce literature data relating to PIP2 presence in interchromatin granule clusters and in the nucleolus, we carried out ultrastructural mapping of PIP2-containing structures using pre-embedding immunolabelling and 3D electron tomography,EELS and super-resolution fluorescence microscopy together with molecular biology experiments. We identified previously nondescribed nucleoplasmic structures decorated by PIP2 molecules, and demonstrated that these structures, referred to as PIP2 islets, are evolutionary conserved. They are surrounded by nucleic acids and protein-containing compounds. At the islet periphery, PIP2 co-localizes with nascent RNA transcripts and the proteins engaged in Pol II transcription and organization of chromatin. PIP2 islets are sensitive to RNase treatment, and their enzymatic disruption affects the level of transcription. Based on our findings, we suggest that PIP2 islets play an important role in the organization of nuclear architecture providing multiple stable surfaces for the formation of the RNA polymerase I/II transcription complexes.
Taken together, this data allowed us to suggest for the first time that PIP2 as a lipid representative plays an important role in the organization of chromatin architecture and in regulation of gene transcription. The results will be discussed in the frame of the current nucleolar model and lipid functions in the cell nucleus.
Permanent Link: http://hdl.handle.net/11104/0270534