Assessment of transferrin recycling by Triplet Lifetime Imaging in living cells

An optical method is presented that allows the measurement of the triplet lifetime of a fluorescent molecule. This is a characteristic specific to each fluorophore. Based on differences in triplet lifetimes of two fluorescent species (autofluorescence versus label), this novel approach measures relative quantities of a transmembrane receptor and associated fluorescently labeled ligand during its recycling in living cells. Similarly to fluorescence-lifetime based methods, our approach is almost insensitive to photobleaching. A simple theory for unmixing two known triplet lifetimes is presented along with validation of the method by measurements of transferrin recycling in a model system based on chinese hamster ovarian cells (CHO). Transferrin is the delivery carrier for Fe3+ to the cell

Temporal Ordering in Endocytic Clathrin-Coated Vesicle Formation via AP2 Phosphorylation.

Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the μ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. μ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with μ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME

Gene Delivery with Hyperbranched Polylysine

The delivery of exogenous nucleic acids into mammalian cells is a valuable technique for basic research studies on the expression, regulation and function of genes and proteins. At the same time, gene delivery to cultivated mammalian cells has become fundamentally important as a technology for the production of therapeutic proteins for preclinical and clinical applications. Alongside with the applications of gene delivery in biotechnology, gene transfer emerged in the early 1990s as a potential therapeutical method in which a functional copy of the defective gene is introduced to provide the missing function. Polymeric cations are widely used as nonviral transfection agents. The efficiency of these vectors, however, is lower than that of viral vectors and has until now precluded their extensive use in applications where stable and high level gene expression is required. Therefore a better understanding of the parameters that govern transfection efficiency of polycationic vectors is crucial for rational design of novel gene carriers. Thus, the main motivation behind this research was to investigate the role of the architecture and molecular weight of the polycation on the efficiency of transfection. The efficiency of transfection is determined by the cytotoxicity of the gene carrier, its reversible complexation with DNA, and proper intracellular trafficking of the resulting complex. For this purpose the 17 member library was synthesized, containing linear (LPL), hyperbranched (HBPL) and dendritic polylysine (DPL) analogues covering a broad range of molecular weights. HBPL, a new class of cationic polymers prepared by polycondensation of L-lysine, was developed to explore the influence of very high molecular weights and randomly branched structures on cytotoxicity, transfection efficiency, and internalization. The thesis is divided into four chapters. Chapter 1 reviews the current state-of-the-art on gene carriers and the future prospects of the field. Additionally, the current use of polycationic gene carriers for the production of recombinant proteins (r-proteins) via a large-scale transient gene expression (TGE) is discussed. The main goal of the Chapter 2 was to determine the influence of the architecture and molecular weight of the polycations on their in vitro biocompatibility. The extent of acute cell death after short exposure was found to be molecular weight dependent. The acute cytotoxicity of high molecular weight polycations was triggered by the permeabilization of the cell membrane. At high concentrations of small molecular weight polycations, membrane destabilization was caused by increased osmotic pressure. In contrast, delayed cell death was correlated to the collapse of mitochondrial transmembrane potential, triggering caspase-dependent apoptosis after intracellular accumulation of the polycation. The onset and extent of the delayed mode of cell death was shown to be dependent on the molecular weight and degree of branching of the polylysine analogues. Based on these results, the differential long term cytotoxicity reflects the resistance of branched polylysine analogues to proteolytic degradation. Thus the degradability and cumulative cytotoxicty can be predetermined by fine-tuning the frequency of branching points in the architecture of peptidic polycation. Chapter 3 demonstrates the evolution of transfection activity with the molecular weight and degree of branching of polylysine analogues. In summary, the results show that under identical cell culture and transfection conditions, the most critical parameter for high gene expression is the structure of the gene carrier. HBPL is the most efficient gene carrier in comparison to LPL and DPL and results in gene expression levels comparable to polyethyleneimine (PEI). Physico-chemical characterization of complexes revealed that those derived from plasmid DNA and HBPL contained a high quantity of the free polycation in comparison to those formed by LPL and DPL. The presence of the free polycation in the course of endocytosis of the complex may provide an explanation for the high transfection efficiency of HBPL. Chapter 4 describes a feasibility study aimed at proving whether HBPL is potentially applicable to large-scale r-protein production via TGE. Gene delivery efficiency and yield of r-protein were evaluated in serum-free, suspension-adapted CHO-DG44 cells under conditions mimicking the industrial manufacture of r-protein by TGE. High molecular weight HBPL mediates transfection and r-protein expression at levels comparable to the ones obtained with PEI. The main advantage of HBPL is its partial enzymatic degradability, potentially decreasing its cumulative cytotoxicity in the course of TGE. Also, HBPL efficiently transfects cell lines and primary cells in the presence of serum, thus broadening the potential applications of HBPL as a novel gene carrier for basic research applications in molecular and cellular biology. In conclusion, it is possible to obtain an efficient gene carrier by simple rearrangement of the topology of poly-L-lysine

Comparative Study on the In Vitro Cytotoxicity of Linear, Dendritic, and Hyperbranched Polylysine Analogues

Lysine-based polycations are widely used as nonviral carriers for gene delivery. This manuscript reports the results of a comparative study on the in vitro cytotoxicity of a library of three structural polylysine variants, namely, linear polylysine (LPL), dendritic polylysine (DPL), and hyperbranched polylysine (HBPL). The aim of this study was to identify possible effects of polymer molecular weight and architecture on both immediate and delayed cytotoxicity and also to provide a mechanistic understanding for possible differences. Acute cytotoxicities were evaluated using cell viability assays with CHO DG44 cells. At comparable molecular weights, the EC50 values for the LPL analogues were similar to 5-250 times higher as compared to the DPL and HBPL samples. For low molecular weight polycations, osmotic shock was found to be an important contributor to immediate cell death, whereas for the higher molecular weight analogues, direct cell membrane disruption was identified to play a role. Delayed cytotoxicity (>= 3 h) was assessed by identifying several of the hallmark events that characterize apoptosis, including phosphatidyl serine translocation, mitochondrial membrane depolarization, cytoplasmic cytochrome C release, and caspase 3 activation. At comparable molecular weights, apoptosis was found to be more pronounced for DPL and HBPL as compared to LPL. This difference was ascribed to the fact that LPL is completely enzymatically degradable, in contrast to DPL and HBPL, which also contain e-peptidic bonds and are only partially degradable. Because their toxicity profiles are similar, HBPL is an interesting (i.e., synthetically easily accessible and inexpensive) alternative to DPL for the nonviral delivery of DNA

FCHO controls AP2’s initiating role in endocytosis through a PtdIns4,5P2 -dependent switch

Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo-adaptor AP2 requires membrane-localised FCHO. Here, live-cell eTIRF-SIM shows that FCHO marks sites of clathrin-coated pit (CCP) initiation, which mature into uniform sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Eps15 ring. We dissect the network of interactions between the FCHO interdomain-linker and AP2, which concentrates, orients, tethers and partially destabilizes closed AP2 at the plasma membrane. AP2’s subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with PtdIns4,5P2, clathrin, cargo and CME accessory factors. FCHO can now relocate toward a CCP's outer edge to engage and activate further AP2s to drive CCP growth/maturation

Regulation of Clathrin-mediated Endocytosis by Hierarchical Allosteric Activation of AP2

The critical initiation phase of clathrin-mediated endocytosis (CME) determines where and when endocytosis occurs. Heterotetrameric adaptor protein 2 (AP2) complexes, which initiate clathrin-coated pit (CCP) assembly, are activated by conformational changes in response to phosphatidylinositol-4,5-bisphosphate (PIP2) and cargo binding at multiple sites. However, the functional hierarchy of interactions and how these conformational changes relate to distinct steps in CCP formation in living cells remains unknown. We used quantitative live-cell analyses to measure discrete early stages of CME and show how sequential, allosterically regulated conformational changes activate AP2 to drive both nucleation and subsequent stabilization of nascent CCPs. Our data establish that cargoes containing Yxxφ motif, but not dileucine motif, play a critical role in the earliest stages of AP2 activation and CCP nucleation. Interestingly, these cargo and PIP2 interactions are not conserved in yeast. Thus, we speculate that AP2 has evolved as a key regulatory node to coordinate CCP formation and cargo sorting and ensure high spatial and temporal regulation of CME

Rapid recombinant protein production from piggyBac transposon-mediated stable CHO cell pools

Heterogeneous populations of stably transfected cells (cell pools) can serve for the rapid production of moderate amounts of recombinant proteins. Here, we propose the use of the piggyBac (PB) transposon system to improve the productivity and long-term stability of cell pools derived from Chinese hamster ovary (CHO) cells. PB is a naturally occurring genetic element that has been engineered to facilitate the integration of a transgene into the genome of the host cell. In this report PB-derived cell pools were generated after 10 days of selection with puromycin. The resulting cell pools had volumetric productivities that were 3–4 times higher than those achieved with cell pools generated by conventional plasmid transfection even though the number of integrated transgene copies per cell was similar in the two populations. In 14-day batch cultures, protein levels up to 600 and 800 mg/L were obtained for an Fc-fusion protein and a monoclonal antibody, respectively, at volumetric scales up to 1 L. In general, the volumetric protein yield from cell pools remained constant for up to 3 months in the absence of selection. In conclusion, transfection of CHO cells with the PB transposon system is a simple, efficient, and reproducible approach to the generation of cell pools for the rapid production of recombinant proteins

Poly(ethyleneimine)-Mediated Large-Scale Transient Gene Expression: Influence of Molecular Weight, Polydispersity and N-Propionyl Groups

Three synthesis lots of linear poly(ethyleneimine) (PEI) are compared to a fully hydrolyzed linear PEI (commercially available as PEI Max) regarding structure, polyplex formation with plasmid DNA, and transfection of suspension-adapted HEK-293E cells. PEI Max binds DNA more efficiently than the other PEIs, but it is the least effective in terms of transient recombinant protein yield. One PEI lot is fractionated by means of SEC. The fractions of high-$\overline {M} _{{\rm n}}$ PEI are the most efficient for complex formation and transfection. Nevertheless, the highest transient recombinant protein yields are achieved with unfractionated PEI. The results demonstrate that the polydispersity and charge density of linear PEI are important parameters for gene delivery to suspension-adapted HEK-293E cells