Fluorescence imaging for monitoring the colocalization of two single molecules in living cells.
AuthorsKoyama-Honda I, Ritchie K, Fujiwara T, Iino R, Murakoshi H, Kasai RS, Kusumi A
JournalBiophys J
PubMed ID15596511
The interaction, binding, and colocalization of two or more molecules in living cells are essential aspects of many biological molecular processes, and single-molecule technologies for investigating these processes in live cells, if successfully developed, would become very powerful tools. Here, we developed simultaneous, dual-color, single fluorescent molecule colocalization imaging, to ... More
Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates.
AuthorsBerlier JE, Rothe A, Buller G, Bradford J, Gray DR, Filanoski BJ, Telford WG, Yue S, Liu J, Cheung CY, Chang W, Hirsch JD, Beechem JM, Haugland RP, Haugland RP
JournalJ Histochem Cytochem
PubMed ID14623938
'Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, ... More
Practical guidelines for dual-color fluorescence cross-correlation spectroscopy.
AuthorsBacia K, Schwille P,
JournalNat Protoc
PubMed ID18007619
'Dual-color fluorescence cross-correlation spectroscopy (FCCS) allows for the determination of molecular mobility and concentrations and for the quantitative analysis of molecular interactions such as binding or cleavage at very low concentrations. This protocol discusses considerations for preparing a biological system for FCCS experiments and offers practical advice for performing FCCS ... More
Virtual and biomolecular screening converge on a selective agonist for GPR30.
AuthorsBologa CG, Revankar CM, Young SM, Edwards BS, Arterburn JB, Kiselyov AS, Parker MA, Tkachenko SE, Savchuck NP, Sklar LA, Oprea TI, Prossnitz ER
JournalNat Chem Biol
PubMed ID16520733
'Estrogen is a hormone critical in the development, normal physiology and pathophysiology of numerous human tissues. The effects of estrogen have traditionally been solely ascribed to estrogen receptor alpha (ERalpha) and more recently ERbeta, members of the soluble, nuclear ligand-activated family of transcription factors. We have recently shown that the ... More
Biologically-active laminin-111 fragment that modulates the epithelial-to-mesenchymal transition in embryonic stem cells.
AuthorsHorejs CM, Serio A, Purvis A, Gormley AJ, Bertazzo S, Poliniewicz A, Wang AJ, Dimaggio P, Hohenester E, Stevens MM,
Journal
PubMed ID24706882
'The dynamic interplay between the extracellular matrix and embryonic stem cells (ESCs) constitutes one of the key steps in understanding stem cell differentiation in vitro. Here we report a biologically-active laminin-111 fragment generated by matrix metalloproteinase 2 (MMP2) processing, which is highly up-regulated during differentiation. We show that the ß1-chain-derived ... More
A transmembrane intracellular estrogen receptor mediates rapid cell signaling.
AuthorsRevankar CM, Cimino DF, Sklar LA, Arterburn JB, Prossnitz ER
JournalScience
PubMed ID15705806
'The steroid hormone estrogen regulates many functionally unrelated processes in numerous tissues. Although it is traditionally thought to control transcriptional activation through the classical nuclear estrogen receptors, it also initiates many rapid nongenomic signaling events. We found that of all G protein-coupled receptors characterized to date, GPR30 is uniquely localized ... More
Size-dependent mechanism of cargo sorting during lysosome-phagosome fusion is controlled by Rab34.
AuthorsKasmapour B, Gronow A, Bleck CK, Hong W, Gutierrez MG,
JournalProc Natl Acad Sci U S A
PubMed ID23197834
'Phagosome maturation is an essential part of the innate and adaptive immune response. Although it is well established that several Ras-related proteins in brain (Rab) proteins become associated to phagosomes, little is known about how these phagosomal Rab proteins influence phagosome maturation. Here, we show a specific role for Rab34 ... More
A novel mechanism for protein delivery: granzyme B undergoes electrostatic exchange from serglycin to target cells.
AuthorsRaja SM, Metkar SS, Höning S, Wang B, Russin WA, Pipalia NH, Menaa C, Belting M, Cao X, Dressel R, Froelich CJ
JournalJ Biol Chem
PubMed ID15788411
The molecular interaction of secreted granzyme B-serglycin complexes with target cells remains undefined. Targets exposed to double-labeled granzyme B-serglycin complexes show solely the uptake of granzyme B. An in vitro model demonstrates the exchange of the granzyme from serglycin to immobilized, sulfated glycosaminoglycans. Using a combination of cell binding and ... More
Podosomes of dendritic cells facilitate antigen sampling.
AuthorsBaranov MV, Ter Beest M, Reinieren-Beeren I, Cambi A, Figdor CG, van den Bogaart G,
Journal
PubMed ID24424029
Dendritic cells sample the environment for antigens and play an important role in establishing the link between innate and acquired immunity. Dendritic cells contain mechanosensitive adhesive structures called podosomes that consist of an actin-rich core surrounded by integrins, adaptor proteins and actin network filaments. They facilitate cell migration via localized ... More
We identified intracellular adhesion molecule-2 (ICAM-2) in a genetic screen as an activator of the PI3K/AKT pathway leading to inhibition of apoptosis. ICAM-2 induced tyrosine phosphorylation of ezrin and PI3K kinase membrane translocation, resulting in phosphatidylinositol 3,4,5 production, PDK-1 and AKT activation, and subsequent phosphorylation of AKT targets BAD, GSK3, ... More
Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. I. Chemical synthesis of various reporter group-labeled 2'-deoxyribonucleoside-5'-triphosphates.
AuthorsGiller G, Tasara T, Angerer B, Mühlegger K, Amacker M, Winter H
JournalNucleic Acids Res
PubMed ID12736313
Fluorescent-labeled DNA is generated through enzymatic incorporation of fluorophore-linked 2'-deoxyribonucleoside-5'-triphosphates (dNTPs) by DNA polymerases. We describe the synthesis of a variety of dye-labeled dNTPs. Amino-linker-modified 5'-triphosphates of all four naturally occurring nucleobases were used as precursors. Commercially available dyes were coupled to the amino function of the side chain. In ... More
Parameters underlying distinct T cell-dependent polysaccharide-specific IgG responses to an intact gram-positive bacterium versus a soluble conjugate vaccine.
AuthorsColino J, Chattopadhyay G, Sen G, Chen Q, Lees A, Canaday DH, Rubtsov A, Torres R, Snapper CM,
JournalJ Immunol
PubMed ID19570830
IgG anti-polysaccharide (PS) responses to both intact Streptococcus pneumoniae (Pn) and PS conjugate vaccines are dependent on CD4(+) T cells, B7-dependent costimulation, and CD40-CD40-ligand interactions. Nevertheless, the former response, in contrast to the latter, is mediated by an ICOS-independent, apoptosis-prone, extrafollicular pathway that fails to generate PS-specific memory. We show ... More
Bovine lactadherin as a calcium-independent imaging agent of phosphatidylserine expressed on the surface of apoptotic HeLa cells.
Bovine lactadherin holds a stereo-specific affinity for phosphatidylserine (PS) membrane domains and binds at PS concentrations lower than the benchmark PS probe, annexin V. Accordingly, lactadherin has recognized PS exposure on preapoptotic immortalized leukemia cells at an earlier time point than has annexin V. In the present study, the cervical ... More
Efficient polyethylenimine-mediated gene delivery proceeds via a caveolar pathway in HeLa cells.
AuthorsGabrielson NP, Pack DW,
JournalJ Control Release
PubMed ID19217921
Most in vivo gene therapies will require cell-specific targeting. Although vector targeting through ligand attachment has met with success in generating gene delivery particles that are capable of specific cellular interactions, little attention has been given to the possible effects of such ligands on subsequent intracellular processing. In this study, ... More
Different transport routes for high density lipoprotein and its associated free sterol in polarized hepatic cells.
AuthorsWüstner D, Mondal M, Huang A, Maxfield FR
JournalJ Lipid Res
PubMed ID14679167
We analyzed the intracellular transport of HDL and its associated free sterol in polarized human hepatoma HepG2 cells. Using pulse-chase protocols, we demonstrated that HDL labeled with Alexa 488 at the apolipoprotein (Alexa 488-HDL) was internalized by a scavenger receptor class B type I (SR-BI)-dependent process at the basolateral membrane ... More
In vivo imaging of germinal centres reveals a dynamic open structure.
AuthorsSchwickert TA, Lindquist RL, Shakhar G, Livshits G, Skokos D, Kosco-Vilbois MH, Dustin ML, Nussenzweig MC
JournalNature
PubMed ID17268470
Germinal centres are specialized structures wherein B lymphocytes undergo clonal expansion, class switch recombination, antibody gene diversification and affinity maturation. Three to four antigen-specific B cells colonize a follicle to establish a germinal centre and become rapidly dividing germinal-centre centroblasts that give rise to dark zones. Centroblasts produce non-proliferating centrocytes ... More
Normal human kidney HLA-DR-expressing renal microvascular endothelial cells: characterization, isolation, and regulation of MHC class II expression.
AuthorsMuczynski KA, Ekle DM, Coder DM, Anderson SK
JournalJ Am Soc Nephrol
PubMed ID12707403
Human, but not murine, renal peritubular and glomerular capillaries constitutively express class II major histocompatibility (MHC) proteins at high levels in normal human kidney. Expression of class II proteins on renal microvascular endothelial cells (RMEC) makes it available to circulating lymphocytes and imparts a surveillance capacity to RMEC for controlling ... More
Dynamic, electronically switchable surfaces for membrane protein microarrays.
AuthorsTang CS, Dusseiller M, Makohliso S, Heuschkel M, Sharma S, Keller B, Vörös J
JournalAnal Chem
PubMed ID16448043
Microarray technology is a powerful tool that provides a high throughput of bioanalytical information within a single experiment. These miniaturized and parallelized binding assays are highly sensitive and have found widespread popularity especially during the genomic era. However, as drug diagnostics studies are often targeted at membrane proteins, the current ... More
Regional loss of the mitochondrial membrane potential in the hepatocyte is rapidly followed by externalization of phosphatidylserines at that specific site during apoptosis.
AuthorsBlom WM, de Bont HJ, Nagelkerke JF
JournalJ Biol Chem
PubMed ID12538597
The spatio-temporal relationship between a decrease in the mitochondrial membrane potential (MMP) and externalization of phosphatidylserines (PS) during induction of apoptosis was investigated in single freshly isolated hepatocytes. Apoptosis was induced in the hepatocytes in three different ways: attack by activated Natural Killer cells, exposure to ATP, or exposure to ... More