Search results for "Fluorescence Recovery after Photobleaching"
showing 10 items of 21 documents
Effect of ATP Binding and Hydrolysis on Dynamics of Canine Parvovirus NS1▿ †
2010
ABSTRACT The replication protein NS1 is essential for genome replication and protein production in parvoviral infection. Many of its functions, including recognition and site-specific nicking of the viral genome, helicase activity, and transactivation of the viral capsid promoter, are dependent on ATP. An ATP-binding pocket resides in the middle of the modular NS1 protein in a superfamily 3 helicase domain. Here we have identified key ATP-binding amino acid residues in canine parvovirus (CPV) NS1 protein and mutated amino acids from the conserved A motif (K406), B motif (E444 and E445), and positively charged region (R508 and R510). All mutations prevented the formation of infectious viruse…
Distribution and Dynamics of Transcription-Associated Proteins during Parvovirus Infection
2012
ABSTRACT Canine parvovirus (CPV) infection leads to reorganization of nuclear proteinaceous subcompartments. Our studies showed that virus infection causes a time-dependent increase in the amount of viral nonstructural protein NS1 mRNA. Fluorescence recovery after photobleaching showed that the recovery kinetics of nuclear transcription-associated proteins, TATA binding protein (TBP), transcription factor IIB (TFIIB), and poly(A) binding protein nuclear 1 (PABPN1) were different in infected and noninfected cells, pointing to virus-induced alterations in binding dynamics of these proteins.
Parvovirus induced alterations in nuclear architecture and dynamics.
2009
The nucleus of interphase eukaryotic cell is a highly compartmentalized structure containing the three-dimensional network of chromatin and numerous proteinaceous subcompartments. DNA viruses induce profound changes in the intranuclear structures of their host cells. We are applying a combination of confocal imaging including photobleaching microscopy and computational methods to analyze the modifications of nuclear architecture and dynamics in parvovirus infected cells. Upon canine parvovirus infection, expansion of the viral replication compartment is accompanied by chromatin marginalization to the vicinity of the nuclear membrane. Dextran microinjection and fluorescence recovery after ph…
Modification of Plasma Membrane Organization in Tobacco Cells Elicited by Cryptogein
2014
Abstract Lipid mixtures within artificial membranes undergo a separation into liquid-disordered and liquid-ordered phases. However, the existence of this segregation into microscopic liquid-ordered phases has been difficult to prove in living cells, and the precise organization of the plasma membrane into such phases has not been elucidated in plant cells. We developed a multispectral confocal microscopy approach to generate ratiometric images of the plasma membrane surface of Bright Yellow 2 tobacco (Nicotiana tabacum) suspension cells labeled with an environment sensitive fluorescent probe. This allowed the in vivo characterization of the global level of order of this membrane, by which w…
Thioflavin T templates amyloid β(1–40) conformation and aggregation pathway
2015
Aβ(1-40) peptide supramolecular assembly and fibril formation processes are widely recognized to have direct implications in the progression of Alzheimer's disease. The molecular basis of this biological process is still unknown and there is a strong need of developing effective strategies to control the occurring events. To this purpose the exploitation of small molecules interacting with Aβ aggregation represents one of the possible routes. Moreover, the use specific labeling has represented so far one of the most common and effective methods to investigate such a process. This possibility in turn rests on the reliability of the probe/labels involved. Here we present evidences of the effe…
Desmosomes: interconnected calcium-dependent structures of remarkable stability with significant integral membrane protein turnover
2002
Desmosomes are prominent cell adhesion structures that are major stabilizing elements, together with the attached cytoskeletal intermediate filament network, of the cytokeratin type in epithelial tissues. To examine desmosome dynamics in tightly coupled cells and in situations of decreased adhesion, fluorescent desmosomal cadherin desmocollin 2a (Dsc2a) chimeras were stably expressed in human hepatocellular carcinoma-derived PLC cells (clone PDc-13) and in Madin-Darby canine kidney cells (clone MDc-2) for the continuous monitoring of desmosomes in living cells. The hybrid polypeptides integrated specifically and without disturbance into normal-appearing desmosomes that occurred in associati…
Polar Localization of a Tripartite Complex of the Two-Component System DcuS/DcuR and the Transporter DctA in Escherichia coli Depends on the Sensor K…
2014
The C4-dicarboxylate responsive sensor kinase DcuS of the DcuS/DcuR two-component system of E. coli is membrane-bound and reveals a polar localization. DcuS uses the C4-dicarboxylate transporter DctA as a co-regulator forming DctA/DcuS sensor units. Here it is shown by fluorescence microscopy with fusion proteins that DcuS has a dynamic and preferential polar localization, even at very low expression levels. Single assemblies of DcuS had high mobility in fast time lapse acquisitions, and fast recovery in FRAP experiments, excluding polar accumulation due to aggregation. DctA and DcuR fused to derivatives of the YFP protein are dispersed in the membrane or in the cytosol, respectively, when …
Non-structural proteins P17 and P33 are involved in the assembly of the internal membrane-containing virus PRD1.
2015
AbstractBacteriophage PRD1, which has been studied intensively at the structural and functional levels, still has some gene products with unknown functions and certain aspects of the PRD1 assembly process have remained unsolved. In this study, we demonstrate that the phage-encoded non-structural proteins P17 and P33, either individually or together, complement the defect in a temperature-sensitive GroES mutant of Escherichia coli for host growth and PRD1 propagation. Confocal microscopy of fluorescent fusion proteins revealed co-localisation between P33 and P17 as well as between P33 and the host chaperonin GroEL. A fluorescence recovery after photobleaching assay demonstrated that the diff…
Identification of Novel Principles of Keratin Filament Network Turnover in Living Cells
2004
It is generally assumed that turnover of the keratin filament system occurs by exchange of subunits along its entire length throughout the cytoplasm. We now present evidence that a circumscribed submembranous compartment is actually the main site for network replenishment. This conclusion is based on the following observations in living cells synthesizing fluorescent keratin polypeptides: 1) Small keratin granules originate in close proximity to the plasma membrane and move toward the cell center in a continuous motion while elongating into flexible rod-like fragments that fuse with each other and integrate into the peripheral KF network. 2) Recurrence of fluorescence after photobleaching i…
Dominance of Chain Entanglement over Transient Sticking on Chain Dynamics in Hydrogen-Bonded Supramolecular Polymer Networks in the Melt
2018
The chain dynamics in supramolecular polymer networks is determined by the interplay of the kinetics of transient interchain association and relaxation of the network chains themselves. This interplay can be addressed by studying model supramolecular polymer networks in which the number of associative side groups and the molar mass of the covalently jointed backbone polymers are both varied systematically. To realize this idea, we use precursor chains with three different molar masses, which comes along with different extents of entanglement in the melt state. For each molar mass, the precursor polymers are functionalized with three different relative contents of associative side groups, gi…