Polycations such as polyethylenimine (PEI) are used in many novel nonviral vector designs and there are continuous efforts to increase our mechanistic understanding of their interactions with cells. Even so, the mechanism of polyplex escape from the endosomal/lysosomal pathway after internalization is still elusive. The ... More
Live cell imaging of G protein-coupled receptors.
AuthorsTeichmann A, Schmidt A, Wiesner B, Oksche A, Schülein R,
JournalMethods Mol Biol
PubMed ID22674164
Live cell imaging experiments with G protein-coupled receptors (GPCRs) tagged with fluorescent fusion proteins were originally performed to study trafficking and subcellular location of these important drug targets. In the past decade, however, substantial progress came from improved imaging methods and from the cloning of novel fluorescent fusion proteins. Today, ... More
Inhibitors of intravesicular acidification protect against Shiga toxin in a pH-independent manner.
AuthorsDyve Lingelem AB, Bergan J, Sandvig K,
JournalTraffic
PubMed ID22132807
Shiga toxin inhibits protein synthesis after being transported from the cell surface to endosomes and retrogradely through the Golgi apparatus to the endoplasmic reticulum (ER) and into the cytosol. In this study, we have abolished proton gradients across internal membranes in different ways and investigated the effect on the various ... More
Engineering of blended nanoparticle platform for delivery of mitochondria-acting therapeutics.
AuthorsMarrache S, Dhar S,
JournalProc Natl Acad Sci U S A
PubMed ID22991470
Mitochondrial dysfunctions cause numerous human disorders. A platform technology based on biodegradable polymers for carrying bioactive molecules to the mitochondrial matrix could be of enormous potential benefit in treating mitochondrial diseases. Here we report a rationally designed mitochondria-targeted polymeric nanoparticle (NP) system and its optimization for efficient delivery of various ... More
Fluorescence lifetime imaging unravels C. trachomatis metabolism and its crosstalk with the host cell.
AuthorsSzaszák M, Steven P, Shima K, Orzekowsky-Schröder R, Hüttmann G, König IR, Solbach W, Rupp J,
JournalPLoS Pathog
PubMed ID21779161
Chlamydia trachomatis is an obligate intracellular bacterium that alternates between two metabolically different developmental forms. We performed fluorescence lifetime imaging (FLIM) of the metabolic coenzymes, reduced nicotinamide adenine dinucleotides [NAD(P)H], by two-photon microscopy for separate analysis of host and pathogen metabolism during intracellular chlamydial infections. NAD(P)H autofluorescence was detected inside ... More
Spreading of neurodegenerative pathology via neuron-to-neuron transmission of ß-amyloid.
Alzheimer's disease (AD) is the major cause of dementia. During the development of AD, neurofibrillary tangles progress in a fixed pattern, starting in the transentorhinal cortex followed by the hippocampus and cortical areas. In contrast, the deposition of ß-amyloid (Aß) plaques, which are the other histological hallmark of AD, does ... More
Baculovirus-mediated gene transfer into mammalian cells.
AuthorsBoyce FM, Bucher NL,
JournalProc Natl Acad Sci U S A
PubMed ID8637876
This paper describes the use of the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) as a vector for gene delivery into mammalian cells. A modified AcMNPV virus was prepared that carried the Escherichia coli lacZ reporter gene under control of the Rous sarcoma virus promoter and mammalian RNA processing ... More
BacMam technology and its application to drug discovery.
AuthorsAmes RS, Kost TA, Condreay JP,
JournalExpert Opin Drug Discov
PubMed ID23488908
The recombinant baculovirus/insect cell system was firmly established as a leading method for recombinant protein production when a new potential use for these viruses was revealed in 1995. It was reported that engineered recombinant baculoviruses could deliver functional expression cassettes to mammalian cell types; a system which has come to ... More
Tools and techniques to measure mitophagy using fluorescence microscopy.
AuthorsDolman NJ, Chambers KM, Mandavilli B, Batchelor RH, Janes MS,
Journal
PubMed ID24121704
Mitophagy is a specialized form of autophagy that removes damaged mitochondria, thereby maintaining efficient cellular metabolism and reducing cellular stress caused by aberrant oxidative bursts. Deficits in mitophagy underlie several diseases, and a substantial body of research has elucidated key steps in the pathways that lead to and execute autophagic ... More
Cellular uptake of cyclotide MCoTI-I follows multiple endocytic pathways.
Cyclotides are plant-derived proteins that naturally exhibit various biological activities and whose unique cyclic structure makes them remarkably stable and resistant to denaturation or degradation. These attributes, among others, make them ideally suited for use as drug development tools. This study investigated the cellular uptake of cyclotide, MCoTI-I in live ... More
IFITM3 directly engages and shuttles incoming virus particles to lysosomes.
AuthorsSpence JS, He R, Hoffmann HH, Das T, Thinon E, Rice CM, Peng T, Chandran K, Hang HC
JournalNat Chem Biol
PubMed ID30643282
'Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) have emerged as important innate immune effectors that prevent diverse virus infections in vertebrates. However, the cellular mechanisms and live-cell imaging of these small membrane proteins have been challenging to evaluate during viral entry of mammalian cells. Using CRISPR-Cas9-mediated IFITM-mutant cell lines, ... More
Importance of measuring endolysosome, cytosolic, and extracellular pH in understanding the pathogenesis of and possible treatments for glioblastoma multiforme.
AuthorsHalcrow P, Khan N, Datta G, Ohm JE, Chen X, Geiger JD
JournalCancer Rep
PubMed ID31989117
'Glioblastoma multiforme (GBM) is a very aggressive form of brain cancer that carries with it a tragically poor prognosis. As with many other forms of cancer, the extracellular environment near GBM tumors is acidified and is relevant to the pathogenesis of GBM because decreased pH promotes tumor cell invasion, increases ... More
Direct Observation of a-Synuclein Amyloid Aggregates in Endocytic Vesicles of Neuroblastoma Cells.
Aggregation of a-synuclein has been linked to both familial and sporadic Parkinson's disease. Recent studies suggest that a-synuclein aggregates may spread from cell to cell and raise questions about the propagation of neurodegeneration. While continuous progress has been made characterizing a-synuclein aggregates in vitro, there is a lack of information ... More
CD44 aptamer mediated cargo delivery to lysosomes of retinal pigment epithelial cells to prevent age-related macular degeneration.
AuthorsChandola C, Casteleijn MG, Chandola UM, Gopalan LN, Urtti A, Neerathilingam M
JournalBiochem Biophys Rep
PubMed ID31080896
Age related macular degeneration (AMD) is a progressive, neurodegenerative disorder that leads to the severe loss of central vision in elderlies. The health of retinal pigment epithelial (RPE) cells is critical for the onset of AMD. Chronic oxidative stress along with loss of lysosomal activity is a major cause for ... More
STF-62247 accumulates in lysosomes and blocks late stages of autophagy to selectively target von Hippel-Lindau-inactivated cells.
AuthorsBouhamdani N, Comeau D, Cormier K, Turcotte S
JournalAm J Physiol Cell Physiol
PubMed ID30758995
Autophagy is a highly conserved, homeostatic process by which cytosolic components reach lysosomes for degradation. The roles played by different autophagic processes in cancer are complex and remain cancer type and stage dependent. Renal cell carcinoma (RCC) is the most common subtype of kidney cancer and is characterized by the ... More