Miroslav Vařecha

How do uncoupling proteins uncouple

According to the proton buffering model, introduced by Klingenberg, UCP1 conducts protons through a hydrophilic pathway lined with fatty acid head groups that buffer the protons as they move across the membrane. According to the fatty acid protonophore model, introduced by Garlid, UCPs do not conduct protons at all. Rather, like all members of this gene family, they are anion carriers. A variety of anions are transported, but the physiological substrates are fatty acid (FA) anions. Because the carboxylate head group is translocated by UCP, and because the protonated FA rapidly diffuses across the membrane, this mechanism permits FA to behave as regulated cycling protonophores. Favoring the latter mechanism is the fact that the head group of long-chain alkylsulfonates, strong acid analogues of FA, is also translocated by UCP.

Bioinformatic and image analyses of the cellular localization of the apoptotic proteins endonuclease G, AIF, and AMID during apoptosis in human cells

We studied the cellular localization of the apoptotic proteins endonuclease G, AIF, and AMID in silico using three prediction tools and in living cells using both single-cell colocalization image analysis and nuclear translocation analysis. We confirmed the mitochondrial localization of endonuclease G and AIF by prediction analysis and by single-cell colocalization image analysis. We found the AMID protein to be cytoplasmic, most probably incorporated into the cytoplasmic side of the membranes of various organelles. The highest concentration of AMID was observed associated with the Golgi. Colocalization of AMID with lysosomes was also indirectly confirmed by analysis of AMID-rich vesicle velocity using manual tracking analysis. Bioinformatic analysis also detected nuclear localization signals in endonuclease G and AIF, but not in AMID. A novel analysis of time-lapse fluorescence image data during staurosporine-induced apoptosis revealed nuclear translocation only for endonuclease G and AIF.

SUV39h- and A-Type Lamin-Dependent Telomere Nuclear Rearrangement

Telomeres are specialized chromatin structures that are situated at the end of linear chromosomes and play an important role in cell senescence and immortalization. Here, we investigated whether changes in histone signature influence the nuclear arrangement and positioning of telomeres. Analysis of mouse embryonic fibroblasts revealed that telomeres were organized into specific clusters that partially associated with centromeric clusters. This nuclear arrangement was influenced by deficiency of the histone methyltransferase SUV39h, LMNA deficiency, and the histone deacetylase inhibitor Trichostatin A (TSA). Similarly, nuclear radial distributions of telomeric clusters were preferentially influenced by TSA, which caused relocation of telomeres closer to the nuclear center. Telomeres also co‐localized with promyelocytic leukemia bodies (PML). This association was increased by SUV39h deficiency and decreased by LMNA deficiency. These differences could be explained by differing levels of the telomerase subunit, TERT, in SUV39h‐ and LMNA‐deficient fibroblasts. Taken together, our data show that SUV39h and A‐type lamins likely play a key role in telomere maintenance and telomere nuclear architecture. J. Cell. Biochem. 109: 915–926, 2010.

Endonuclease G interacts with histone H2B and DNA topoisomerase II alpha during apoptosis

Apoptosis is a natural form of cell death involved in many physiological changes in the cell. Defects in the process of apoptosis can lead to serious diseases. During some apoptotic pathways, proteins apoptosis-inducing factor (AIF) and endonuclease G (EndoG) are released from the mitochondria and they translocate into the cell nuclei, where they probably participate in chromatin degradation together with other nuclear proteins. Exact mechanism of EndoG activity in cell nucleus is still unknown. Some interacting partners like flap endonuclease 1, DNase I, and exonuclease III were already suggested, but also other interacting partners were proposed. We conducted a living-cell confocal fluorescence microscopy followed by an image analysis of fluorescence resonance energy transfer to analyze the possibility of protein interactions of EndoG with histone H2B and human DNA topoisomerase II alpha (TOPO2a). Our results show that EndoG interacts with both these proteins during apoptotic cell death. Therefore, we can conclude that EndoG and TOPO2a may actively participate in apoptotic chromatin degradation. The possible existence of a degradation complex consisting of EndoG and TOPO2a and possibly other proteins like AIF and cyclophilin A have yet to be investigated.

An inactivating mutation in intestinal cell kinase, ICK, impairs hedgehog signalling and causes short rib-polydactyly syndrome.

The short rib polydactyly syndromes (SRPS) are a group of recessively inherited, perinatal-lethal skeletal disorders primarily characterized by short ribs, shortened long bones, varying types of polydactyly and concomitant visceral abnormalities. Mutations in several genes affecting cilia function cause SRPS, revealing a role for cilia function in skeletal development. To identify additional SRPS genes and discover novel ciliary molecules required for normal skeletogenesis, we performed exome sequencing in a cohort of patients and identified homozygosity for a missense mutation, p.E80K, in Intestinal Cell Kinase, ICK, in one SRPS family. The p.E80K mutation abolished serine/threonine kinase activity, resulting in altered ICK subcellular and ciliary localization, increased cilia length, aberrant cartilage growth plate structure, defective Hedgehog and altered ERK signalling. These data identify ICK as an SRPS-associated gene and reveal that abnormalities in signalling pathways contribute to defective skeletogenesis.

Prediction of localization and interactions of apoptotic proteins

During apoptosis several mitochondrial proteins are released. Some of them participate in caspase-independent nuclear DNA degradation, especially apoptosis-inducing factor (AIF) and endonuclease G (endoG). Another interesting protein, which was expected to act similarly as AIF due to the high sequence homology with AIF is AIF-homologous mitochondrion-associated inducer of death (AMID). We studied the structure, cellular localization, and interactions of several proteins in silico and also in cells using fluorescent microscopy. We found the AMID protein to be cytoplasmic, most probably incorporated into the cytoplasmic side of the lipid membranes. Bioinformatic predictions were conducted to analyze the interactions of the studied proteins with each other and with other possible partners. We conducted molecular modeling of proteins with unknown 3D structures. These models were then refined by MolProbity server and employed in molecular docking simulations of interactions. Our results show data acquired using a combination of modern in silico methods and image analysis to understand the localization, interactions and functions of proteins AMID, AIF, endonuclease G, and other apoptosis-related proteins.

Stage-specific roles of FGF2 signaling in human neural development

This study elucidated the stage-specific roles of FGF2 signaling during neural development using in-vitro human embryonic stem cell-based developmental modeling. We found that the dysregulation of FGF2 signaling prior to the onset of neural induction resulted in the malformation of neural rosettes (a neural tube-like structure), despite cells having undergone neural induction. The aberrant neural rosette formation may be attributed to the misplacement of ZO-1, which is a polarized tight junction protein and shown co-localized with FGF2/FGFR1 in the apical region of neural rosettes, subsequently led to abnormal neurogenesis. Moreover, the FGF2 signaling inhibition at the stage of neural rosettes caused a reduction in cell proliferation, an increase in numbers of cells with cell-cycle exit, and premature neurogenesis. These effects may be mediated by NUMB, to which expression was observed enriched in the apical region of neural rosettes after FGF2 signaling inhibition coinciding with the disappearance of PAX6+/Ki67+ neural stem cells and the emergence of MAP2+ neurons. Moreover, our results suggested that the hESC-based developmental system reserved a similar neural stem cell niche in vivo.

Optical tracking of micro-objects within living cells

Tracing of foreign objects inside living cells is very exciting way how to study interior of living objects in nondestructive way. We imported fluorescent submicron particles into the living cells using liposomes as carriers to study the local mechanical heterogeneity of the cell cytoplasm. Thermal motion of these probes within the cell is tracked using fluorescent video-microscopy. The time-records of the probe positions reveal their trajectories and accessible space to the probes inside the cytoplasm of living cells. Further analyses of the thermal motion of the probes can reveal the mechanism of sub-cellular transport and properties of the cytoplasm in vivo.