Since the recent boost in the usage of electron microscopy in life-science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused-ion beam scanning electron microscopy (FIB-SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold-labelled breast cancer SKBR3 cells was to visualise gold-labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 μm2 cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back-scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB-SEM. Cross-sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms.

Immuno-electron microscopy is commonly performed with the use of antibodies. In the last decade the antibody fragment indicated as nanobody (VHH or single domain antibody) has found its way to different applications previously done with conventional antibodies. Nanobodies can be selected to bind with high affinity and specificity to different antigens. They are small (molecular weight ca. 15kDa) and are usually easy to produce in microorganisms. Here we have evaluated the feasibility of a nanobody binding to HER2 for application in immuno-electron microscopy. To obtain highest labeling efficiency combined with optimal specificity, different labeling conditions were analysed, which included nanobody concentration, fixation and blocking conditions. The obtained optimal protocol was applied for post-embedment labeling of Tokuyasu cryosections and for pre-embedment labeling of HER2 for fluorescence microscopy and both transmission and scanning electron microscopy. We show that formaldehyde fixation after incubation with the anti-HER2 nanobody, improves labeling intensity. Among all tested blocking agents the best results were obtained with a mixture of cold water fish gelatine and acetylated bovine serum albumin, which prevented a-specific interactions causing background labeling while preserving specific interactions at the same time. In conclusion, we have developed a nanobody-based protocol for immuno-gold labeling of HER2 for Tokuyasu cryosections in TEM as well as for pre-embedment gold labeling of cells for both TEM and SEM.

The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase.

Amino acid permeases (AAPs) in the plasma membrane (PM) of Saccharomyces cerevisiae are responsible for the uptake of amino acids and involved in regulation of their cellular levels. Here, we report on a strong and complex module for PM association found in the C-terminal tail of AAPs. Using in silico analyses and mutational studies we found that the C-terminal sequences of Gap1, Bap2, Hip1, Tat1, Tat2, Mmp1, Sam3, Agp1, and Gnp1 are about 50 residues long, associate with the PM, and have features that discriminate them from the termini of organellar amino acid transporters. We show that this sequence (named PMasseq) contains an amphipathic alpha-helix and the FWC signature, which is palmitoylated by palmitoyltransferase Pfa4. Variations of PMasseq, found in different AAPs, lead to different mobilities and localization patterns, whereas the disruption of the sequence has an adverse effect on cell viability. We propose that PMasseq modulates the function and localization of AAPs along the PM. PMasseq is one of the most complex protein signals for plasma membrane association across species and can be used as a delivery vehicle for the PM.

Protein molecules, toxins and viruses internalize into the cell via receptor-mediated endocytosis (RME) using specific proteins and lipids in the plasma membrane. The plasma membrane is a barrier for many pharmaceutical agents to enter into the cytoplasm of target cells. In the case of cancer cells, tissue-specific biomarkers in the plasma membrane, like cancer-specific growth factor receptors, could be excellent candidates for RME-dependent drug delivery. Recent data suggest that agent binding to these receptors at the cell surface, resulting in membrane domain formation by receptor clustering, can be used for the initiation of RME. As a result, these pharmaceutical agents are internalized into the cells and follow different routes until they reach their final intracellular targets like lysosomes or Golgi. We propose that clustering induced formation of plasma membrane microdomains enriched in receptors, sphingolipids, and inositol lipids, leads to membrane bending which functions as the onset of RME. In this review we will focus on the role of domain formation in RME and discuss potential applications for targeted intracellular drug delivery.

The TIM23 complex mediates translocation of proteins across, and their lateral insertion into, the mitochondrial inner membrane. Translocation of proteins requires both the membrane-embedded core of the complex and its ATP-dependent import motor. Insertion of some proteins, however, occurs in the absence of ATP, questioning the need for the import motor during lateral insertion. We show here that the import motor associates with laterally inserted proteins even when its ATPase activity is not required. Furthermore, our results suggest a role for the import motor in lateral insertion. Thus, the import motor is involved in ATP-dependent translocation and ATP-independent lateral insertion.

It is well known that breast tumors which are estrogen positive ER(+) are more likely to respond to hormone therapy. However, a certain percentage of ER(+)/PR(+) tumors do not respond to this therapy. Identification of the second estrogen receptor, named estrogen receptor beta (ER?), as well as the existence of numerous isoforms/splice variants of both ER? and ER?, suggests that a complex regulation of estrogen action exists. In this study, we analyzed the expression ratio of ER?1 isoform and ER??5 splice variant mRNAs, and its correlation with ER/PR status by quantitative RT-PCR and clinical and histopathological parameters. We found that the relative proportion of ER??5 in the total ER?1 transcript 'pool' inversely correlates with the PR level (p = -0,359, p< 0,003, Spearman). It may be that the ER??5 variant modulates the ER? activity of downstream targets. In addition, we suggest that the determination of the expression profiles of ER? and ER? isoforms and splice variants in the defined groups of patients are necessary for elucidating their involvement in endocrine resistance.

Transport of essentially all matrix and a number of inner membrane proteins is governed, entirely or in part, by N-terminal presequences and requires a coordinated action of the translocases of outer and inner mitochondrial membranes (TOM and TIM23 complexes). Here, we have analyzed Tim50, a subunit of the TIM23 complex that is implicated in transfer of precursors from TOM to TIM23. Tim50 is recruited to the TIM23 complex via Tim23 in an interaction that is essentially independent of the rest of the translocase. We find Tim50 in close proximity to the intermembrane space side of the TOM complex where it recognizes both types of TIM23 substrates, those that are to be transported into the matrix and those destined to the inner membrane, suggesting that Tim50 recognizes presequences. This function of Tim50 depends on its association with TIM23. We conclude that the efficient transfer of precursors between TOM and TIM23 complexes requires the concerted action of Tim50 with Tim23.

The TIM23 complex is the major translocase of the mitochondrial inner membrane responsible for the import of essentially all matrix proteins and a number of inner membrane proteins. Tim23 and Tim50, two essential proteins of the complex, expose conserved domains into the intermembrane space that interact with each other. Here, we describe in vitro reconstitution of this interaction using recombinantly expressed and purified intermembrane space domains of Tim50 and Tim23. We established two independent methods, chemical cross-linking and surface plasmon resonance, to track their interaction. In addition, we identified mutations in Tim23 that abolish its interaction with Tim50 in vitro. These mutations also destabilized the interaction between the two proteins in vivo, leading to defective import of preproteins via the TIM23 complex and to cell death at higher temperatures. This is the first study to describe the reconstitution of the Tim50-Tim23 interaction in vitro and to identify specific residues of Tim23 that are vital for the interaction with Tim50.

The TIM23 (translocase of the mitochondrial inner membrane) complex mediates translocation of preproteins across and their insertion into the mitochondrial inner membrane. How the translocase mediates sorting of preproteins into the two different subcompartments is poorly understood. In particular, it is not clear whether association of two operationally defined parts of the translocase, the membrane-integrated part and the import motor, depends on the activity state of the translocase. We established conditions to in vivo trap the TIM23 complex in different translocation modes. Membrane-integrated part of the complex and import motor were always found in one complex irrespective of whether an arrested preprotein was present or not. Instead, we detected different conformations of the complex in response to the presence and, importantly, the type of preprotein being translocated. Two non-essential subunits of the complex, Tim21 and Pam17, modulate its activity in an antagonistic manner. Our data demonstrate that the TIM23 complex acts as a single structural and functional entity that is actively remodelled to sort preproteins into different mitochondrial subcompartments.

Mandušić, Vesna; Krtolica-Žikić, Koviljka; Nikolić-Vukosavljević, Dragica; Popov-Čeleketić, D.; Plećaš, D.; Boričić, I.; Dimitrijević, B.; Tanić, N. - Transcriptional ratio of estrogen receptor β mRNAs in carcinomas and in normal tissues - Archives of Biological Sciences

Translocation of preproteins with N-terminal presequences into mitochondria requires the cooperation of the translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex). However, the molecular nature of the translocation contact sites is poorly understood. We have identified a novel component of the TIM23 translocase, Tim21, which is involved in their formation. Tim21 is anchored in the mitochondrial inner membrane by a single transmembrane domain and exposes its C-terminal domain into the intermembrane space. The purified C-terminal domain of Tim21 appears not to bind to any of the TIM23 components but rather specifically interacts with the TOM complex. We propose that Tim21 binds to the trans site of the TOM complex thus keeping the two translocases in close contact.

Tiazofurin (TZF) is a cytostatic drug that leads to depletion of the GTP pool in tumor and normal cells via its active metabolite tiazofurin-adenine dinucleotide (TAD). TAD was detected in different cell lines, but not in erythrocytes, so the mechanism of erythrocytotoxicity of TZF remains unclear. The purpose of this study was to evaluate in vitro and in vivo action of tiazofurin on rat erythrocytes (RBC). After two decades of clinical trials the question of erythrocytotoxicity of TZF had remained unexplained making this study justified. Since we have previously demonstrated early erythrocytotoxic effects in male Wistar rats, we extend this finding on isolated RBC. Isolated erythrocytes from untreated animals were treated in buffered solution or plasma containing TZF. In addition, groups of 10 rats were treated with 200 and 1000 mg/kg of TZF and hematologic parameters were analyzed by flowcytometry and by the analysis of the peripheral blood smears. Early signs of hemolysis or aberrant structures were monitored by scanning probe microscopy (SPM). We suggest that correlation exists between early erythrocytotoxicity and irregularities in erythrocyte morphology and membrane integrity. We also found that TZF affects responsiveness to oxidative stress. This is in concordance with flowcytometric findings describing anisocytosis and anisochromosis of RBC.