Dynamic Regulation of Function of the Mitochondrial TIM23 Preprotein Translocase

The vast majority of mitochondrial proteins are synthesized on cytosolic ribosomes in the form of precursor proteins and subsequently imported into mitochondria through the concerted action of the translocases present in the outer and the inner membrane. The TIM23 complex (translocase of the inner membrane) mediates translocation of precursor proteins across or their insertion into the mitochondrial inner membrane in a membrane potential and ATP-dependent manner. The TIM23 complex consists of eight essential subunits that can be assigned to two operationally defined parts: the membrane embedded protein conducting channel with the receptor and the import motor associated with the channel at the matrix side of the inner membrane. The present study was undertaken to gain insight into the dynamics of the TIM23 translocase during import of different types of preproteins. A previously uncharacterized protein component of the TIM23 translocase was identified and termed Tim21. Results presented in this study demonstrate that the TIM23 translocase switches between translocation mode that facilitates import of proteins into the matrix and insertion mode that allows lateral sorting of proteins into the lipid bilayer. The TIM23 translocase adopts different conformations in its various states of activity: when it was empty, when it inserted preproteins into the inner membrane and when it translocated preproteins targeted to the matrix. The interconversion of the TIM23 translocase between the functional states occurs primarily by conformational changes of the essential components, whereas non-essential components Tim21 and Pam17 are responsible for the fine tuning of these processes. A hypothesis that describes the behavior of the TIM23 translocase is presented

Shape invariant potential formalism for photon-added coherent state construction

An algebro-operator approach, called shape invariant potential method, of constructing generalized coherent states for photon-added particle system is presented. Illustration is given on Poschl-Teller potential

Chemical Mapping of Ceramide Distribution in Sphingomyelin-Rich Domains in Monolayers

The incorporation of ceramide in phase-separated monolayers of ternary lipid mixtures has been studied by a combination of atomic force microscopy (AFM), fluorescence, and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Replacement of a fraction of the sphingomyelin by ceramide in DOPC/SM/cholesterol monolayers leads to changes in the SM-cholesterol-rich liquid-ordered domains. AFM shows the formation of heterogeneous domains with small raised islands that are assigned to a ceramide-rich gel phase. ToF-SIMS provides conclusive evidence for the localization of SM and ceramide in ordered domains and shows that ceramide is heterogeneously distributed in small islands throughout the domains. The results indicate the utility of combining AFM and ToF-SIMS for understanding compositions of phase-separated membranes

Some Theoretical Observations Concerning the Reverberation Time for the Case of a Harmonic Emitting Source

In the paper, the reverberation phenomenon produced by the harmonic emission of a sound source is theoretically examined, taking into consideration the absorption of the sound in the air. We obtain a formula which contains, as particular cases, the similar formulae presented in the literature

Membrane domain formation - a key factor for targeted intracellular drug delivery

Proteinmolecules,toxinsandvirusesinternalizeintothecellviareceptor-mediatedendocytosis(RME)usingspecificproteinsandlipidsintheplasmamembrane.Theplasmamembraneisabarrierformanypharmaceuticalagentstoenterintothecytoplasmoftargetcells.Inthecaseofcancercells,tissue-specificbiomarkersintheplasmamembrane,likecancer-specificgrowthfactorreceptors,couldbeexcellentcandidatesforRME-dependentdrugdelivery.Recentdatasuggestthatagentbindingtothesereceptorsatthecellsurface,resultinginmembranedomainformationbyreceptorclustering,canbeusedfortheinitiationofRME.Asaresult,thesepharmaceuticalagentsareinternalizedintothecellsandfollowdifferentroutesuntiltheyreachtheirfinalintracellulartargetslikelysosomesorGolgi.Weproposethatclusteringinducedformationofplasmamembranemicrodomainsenrichedinreceptors,sphingolipids,andinositollipids,leadstomembranebendingwhichfunctionsastheonsetofRME.InthisreviewwewillfocusontheroleofdomainformationinRMEanddiscusspotentialapplicationsfortargetedintracellulardrugdelivery

Nucleolus, The ribosome factory

The nucleolus is a nuclear compartment and represents the most obvious and clearly differentiated nuclear structure seen in the microscope. Within nucleoli most events of ribosome biogenesis, such as ribosomal RNA synthesis, processing, and ribosome subunit assembly, take place. Several lines of evidence now show that the nucleolus has also numerous nonribosomal functions. This review is focused on the recent progress in our knowledge of how to correlate the known biochemical processes taking place in the nucleolus with nucleolar structures observed in the microscope. We still lack detailed enough information to understand fully the organization and regulation of the processes taking place in the nucleolar sub-structures. However, the present power of microscopy techniques should allow for an in situ description of the organization of nucleolar processes at the molecular level in the years to come

A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy

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

A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy

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

EGFR Dynamics Change during Activation in Native Membranes as Revealed by NMR

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