The centre of the cell
Image: Unravelling the cell nucleus; Pavel Hozak and his team at the Laboratory of Biology of the Cell Nucleus.
When a team of researchers from the Czech Republic and US, discovered that actin-binding molecular motor proteins existed in the nucleus of a cell, biologists were staggered.
Filaments are central to the biological functions of actins and while widely detected in cytoplasm, little trace of these structures in the cell's control centre left researchers convinced that nuclear actin and myosin simply didn't exist.
However, by using fluorescence, confocal and immunoelectron microscopy, molecular biologist, Dr Pavel Hozák, and fellow researchers from the Academy of Sciences of the Czech Republic and Universities of Illinois at Chicago and Virginia, disbanded this myth.
Their correlative microscopy studies plainly showed the actin-based molecular motor, Myosin I, within mouse embryo cells. And as they stated at the time in Science: "Data suggests the nuclear myosin I, perhaps by interacting with RNA polymerases, could potentially power transcription."
For Hozák, now Group Leader of the Laboratory of Biology of the Cell Nucleus at the Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, location was, and always will be, critical to cell studies.
"I have always thought that mechanistically studying biological events is not enough; we also need to see where the event is taking place and which molecules are present."
In the run up to the nuclear actin and myosin breakthrough, the young researcher had spent several years at the Sir William Dunn School of Pathology, University of Oxford, working with Professor Peter Cook on nuclear structure and function.
Access to, at the time, cutting-edge confocal and cryo-electron microscopes was fuelling their research into the foci within cell nuclei, responsible for replicating genetic information. The pair had also developed a thick sectioning sample preparation method so they could look at cellular networks within a nuclei.
"We could observe these foci and describe their substructures, for the first time, using electron microscopy," highlights Hozák. "And the sectioning technique meant we could view [a complete] intra-nuclei network in 3D."
The genetic discoveries that ensued shook the foundations of established DNA dynamics understanding, but come the late 1990s, Hozák had made his move to Prague to set up the Laboratory of Biology of the Cell Nucleus at the Institute of Experimental Medicine, and later at the Institute of Molecular Genetics.
At the time, Hozák was working with two students and a technician, and was interested in developing algorithms to better detect and evaluate the metallic nanoparticles used for labelling in immuno-electron microscopy.
His team developed programs to determine co-localisation and clustering patterns that could be applied to nanoparticle populations, proving crucial to their nuclear actin and myosin breakthroughs. And as Hozák highlights: "This really kick-started our new area of research in the lab and really helped us to discover the spectrum of actin-binding proteins in the cell nucleus."
Structured illumination microscopy of lipids involved in ribosomal RNA synthesis.
Come 2004, Hozák alongside his growing team at the Laboratory of Biology of the Cell Nucleus as well as colleagues from the German Cancer Research Center and University of Illinois at Chicago, had proven that nuclear actin and myosin I existed and were required for RNA transcription, as detailed in Nature Cell Biology.
Research into nuclear actin and myosin continued but Hozák and his team were also keen to develop new nanoparticles for multiple immuno-labelling in electron microscopy.
The use of gold nanoparticles tagged with immunoglobulin was limited to two targets as the nanoparticles were only distinguishable by size. But as Hozák and many others were aware, increasing the number of targets that could be identified simultaneously would shed more light on important molecular localisation and interactions.
"This was a major problem so we joined forces with researchers from the Chemistry Department here in Prague to develop new colloidal nanoparticles that were also different in shape," he explains.
To this end, the researchers developed a mix of cubic palladium nanoparticles, gold nanorods and gold-silver core-shell nanoparticles, and used these to map the localisation of a nuclear lipid phosphatidylinositol alongside four other molecules critical to genome function.
Results were published in Histochemistry and Cell Biology in 2014, and as Hozák asserts: "This was the first time in ultrastructural histochemistry, that up to five molecular targets had been simultaneously identified."
According to Hozák, he and his colleagues' methodological advances in sample preparation, computer image analysis and immuno-labelling have been fundamental to research progress.
His laboratory now comprises more than ten researchers and three technicians, currently working with myriad grants on actin-related proteins, nuclear lamins, phosphoinositide compartments in the cell nucleus and more.
As part of their research into nuclear lipids, the team revealed the true role of these phosphoinositides in DNA transcription. As Hozák highlights: "It was originally thought that the reading of genetic information in the cell nucleus was regulated by proteins and polymerases but we have discovered that lipids can participate as well."
Pavel Hozak: "We really need to observe individual molecules without attaching them to a tag; having this would be absolutely amazing."
Using super-resolution, electron microscopy and molecular methods, the team showed that lipids can modify the conformation of proteins that regulate gene transcription.
And as Hozák highlights: "Microscopy has helped us enormously and a combination of super resolution microscopy and electron microscopy has been crucial."
"Using these methods, we were, for example, able to show previously undescribed lipid-containing structures in the cell nucleus and that surface transcription can occur on the surface," he adds.
For this latest research, Hozák and colleagues used the FEI Tecnai TEM and Gatan energy filter as well as 3D Structured Illumination and Dense Stochastic Sampling Imaging - Localization Microscopy from GE Healthcare or Nikon, and 3D Gated Stimulated Emission Depleted Microscopy from Leica.
And along the way, Hozák's team has closely collaborated with microscopy giants, FEI and TESCAN. Projects with FEI proved instrumental to the team's breakthroughs in sample processing and algorithm development.
Meanwhile, the team has been testing TESCAN's recently developed multi-modal holographic microscope, Q-PHASE, based on coherence-controlled holographic microscopy for quantitative phase imaging.
"We were looking at high throughput analysis here," says Hozák. "Holographic microscopes can better detect the edges of cell and this instrument proved to be much more convenient than standard microscopy techniques such as phase contrast microscopy."
Beyond the Laboratory of Biology of the Cell Nucleus, Hozák also heads up the Microscopy Centre at the Institute of Molecular Genetics, which comprises the light microscopy and electron microscopy divisions.
The Light Microscopy division includes Leica confocal and fluorescence microscopes, Leica STED and GE Healthcare SIM super-resolution microscopes.
Meanwhile, the electron microscopy facility comprises two TEMs; a FEI Morgagni 268 for routine observation and a 200 kV FEI Tecnai G2 20 LaB6 for high-resolution TEM, 3D electron tomography, cryo-EM and electron energy-loss (EELS) analysis. Additional instrumentation includes Leica and FEI cryo-EM sample preparation equipment and Leica (cryo)ultramicrotomes.
Structured illumination microscopy of lipid-containing structures involved in reading genetic information.
Crucially, the Microscopy Centre now also serves as a hub for 'Czech Bioimaging' a national research infrastructure for biological and medical imaging that consists of seven imaging facilities in the Czech Republic.
"Our new network started in January this year and will provide open access to the newest technologies in microscopy for researchers from the Czech Republic and abroad," points out Hozák.
Importantly for researchers, Czech Bioimaging has also joined a pan-European biological and medical imaging initiative, 'Euro-Bioimaging'.
From confocal, multi-photon, total internal reflection fluorescence microscopy and STED to fluorescence resonance energy transfer, Raman spectroscopy, electron microscopy and more, the breadth of imaging technologies is staggering. And the Microscopy Centre is one of only two hosting locations in the Czech Republic.
"With this high level of expertise we hope to achieve faster research progress in biomedicine," says Hozák. "It will be wonderful as we can really provide the infrastructure to facilitate and advance research; it's so exciting."
Hozák believes a wider access to a range of super-resolution microscopy methods in combination with structural biology techniques will drive breakthroughs in studies of structures smaller than 100 nm.
As he points out: "This is relevant to most biological processes that occur in macromolecule complexes; in the past we could have only guessed what happened but now we can observe, for example, how molecules move inside these complexes, even in living cells."
Looking to the future, Hozák, like many, is certain that super-resolution methods will be crucial to research into living cells, especially studying cell dynamics. He would also like to see Raman microscopy at super-resolution without the use of fluorescent tags as well as better links between microscopy and mass spectroscopy imaging.
"Instruments that connect imaging with mass spectroscopy are already underway but now we need better precision and resolution for the new histochemistry," he says. " And we really need to observe individual molecules without attaching them to a tag; having this would be absolutely amazing."