Polymer film-based microwell array platform for long-term culture and research of human bronchial organoids.

The culture of lung organoids relies on drops of basement membrane matrices. This comes with limitations, for example, concerning the microscopic monitoring and imaging of the organoids in the drops. Also, the culture technique is not easily compatible with micromanipulations of the organoids. In this study, we investigated the feasibility of the culture of human bronchial organoids in defined x-, y- and z-positions in a polymer film-based microwell array platform. The circular microwells have thin round/U-bottoms. For this, single cells are first precultured in drops of basement membrane extract (BME). After they form cell clusters or premature organoids, the preformed structures are then transferred into the microwells in a solution of 50% BME in medium. There, the structures can be cultured toward differentiated and mature organoids for several weeks. The organoids were characterized by bright-field microscopy for size growth and luminal fusion over time, by scanning electron microscopy for overall morphology, by transmission electron microscopy for the existence of microvilli and cilia, by video microscopy for beating cilia and swirling fluid, by live-cell imaging, by fluorescence microscopy for the expression of cell-specific markers and for proliferating and apoptotic cells, and by ATP measurement for extended cell viability. Finally, we demonstrated the eased micromanipulation of the organoids in the microwells by the example of their microinjection.

Synergistic and bactericidal activities of mecillinam, amoxicillin and clavulanic acid combinations against extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in 24-h time-kill experiments.

This study aimed to evaluate the potential synergistic and bactericidal effects of mecillinam in combination with amoxicillin and clavulanic acid against extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli. Eight clinical E. coli isolates with varying susceptibility to mecillinam [minimum inhibitory concentrations (MICs) of 0.125 mg/L to >256 mg/L] and high-level resistance to amoxicillin (MICs > 256 mg/L) were used. Whole-genome sequencing was performed to determine the presence of ß-lactamase genes and mutations in the cysB gene. The activities of single drugs and the combinations of two or three drugs were tested in 24-h time-kill experiments. Population analysis was performed for two strains before and after experiments. Only one strain had a mutation in the cysB gene resulting in an amino acid substitution. With the two-drug combinations, initial killing was observed both with mecillinam and amoxicillin when combined with clavulanic acid. Synergy was observed with mecillinam and clavulanic acid against one strain and with amoxicillin and clavulanic acid against three strains. However, following significant re-growth, a bactericidal effect was found only with amoxicillin and clavulanic acid against two strains. Pre-existing subpopulations with elevated mecillinam MICs were detected before experiments and were selected with mecillinam alone or in two-drug combinations. In contrast, the three-drug combination showed enhanced activity with synergy against six strains, a bactericidal effect against all eight strains, and suppression of resistance during 24-h antibiotic exposure. This combination may be of clinical interest in the treatment of urinary tract infections caused by ESBL-producing E. coli.

Bimolecular fluorescence complementation (BiFC) technique in yeast Saccharomyces cerevisiae and mammalian cells.

Visualization of protein-protein interactions in vivo offers a powerful tool to resolve spatial and temporal aspects of cellular functions. The bimolecular fluorescence complementation (BiFC) makes use of nonfluorescent fragments of green fluorescent protein or its variants that are added as "tags" to target proteins under study. Only upon target protein interaction is a fluorescent protein complex assembled, and the site of interaction can be monitored by microscopy. In this chapter, we describe the method and tools for the use of BiFC in the yeast Saccharomyces cerevisiae and in mammalian cells.

Actin as a model for the study of nucleocytoplasmic shuttling and nuclear dynamics.

A great number of molecules are constantly being exchanged between the nucleus and the cytoplasm via nuclear pore complexes (NPCs). Importantly, this nucleocytoplasmic trafficking is used to transfer information between the two compartments, thereby permitting the manipulation of critical nuclear processes such as transcription. Constant shuttling of actin is an example of the versatility of this regulatory avenue, as this protein has the capability to drive the transcriptional activity of certain gene sets as well as influence transcription on a global scale. Nuclear import and export are extremely dynamic phenomena and require imaging tools capable of rapid sampling rates for proper quantitative observation. Here we describe live-cell imaging assays based on fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) for monitoring both import and export of fluorescently labelled molecules. Our assays are performed with GFP-actin, but the same principle is applicable to most proteins shuttling between the nucleus and the cytoplasm. Furthermore, these assays may also expose novel qualities of the intranuclear dynamics of a protein, which can polymerize or partake in complexes, because such behavior is mirrored in the nuclear retention of the protein detectable by both import and export assays.

Steady-state nuclear actin levels are determined by export competent actin pool.

A number of studies in the last decade have irrevocably promoted actin into a fully fledged member of the nuclear compartment, where it, among other crucial tasks, facilitates transcription and chromatin remodeling. Changes in nuclear actin levels have been linked to different cellular processes: decreased nuclear actin to quiescence and increased nuclear actin to differentiation. Importin 9 and exportin 6 transport factors are responsible for the continuous nucleocytoplasmic shuttling of actin, but the mechanisms, which result in modulated actin levels, have not been characterized. We find that in cells growing under normal growth conditions, the levels of nuclear actin vary considerably from cell to cell. To understand the basis for this, we have extensively quantified several cellular parameters while at the same time recording the import and export rates of green fluorescent protein (GFP)-tagged actin. Surprisingly, our dataset shows that the ratio of nuclear to cytoplasmic fluorescence intensity, but not nuclear shape, size, cytoplasm size, or their ratio, correlates negatively with both import and export rate of actin. This suggests that high-nuclear actin content is maintained by both diminished import and export. The high nuclear actin containing cells still show high mobility of actin, but it is not export competent, suggesting increased binding of actin to nuclear complexes. Creation of such export incompetent actin pool would ensure enough actin is retained in the nucleus and make it available for the various nuclear functions described for actin.

Actin-regulated feedback loop based on Phactr4, PP1 and cofilin maintains the actin monomer pool.

Phactr proteins bind actin and protein phosphatase 1 (PP1), and are involved in processes ranging from angiogenesis to cell cycle regulation. Phactrs share a highly conserved RPEL domain with the myocardin-related transcription factor (MRTF) family, where actin binding to this domain regulates both the nuclear localization and the activity of these transcription coactivators. We show here that in contrast to MRTF-A, the RPEL domain is dispensable for the subcellular localization of Phactr4. Instead, we find the domain facilitating competitive binding of monomeric actin and PP1 to Phactr4. Binding of actin to Phactr4 influences the activity of PP1 and the phosphorylation status of one of its downstream targets, cofilin. Consequently, at low actin monomer levels, Phactr4 guides PP1 to dephosphorylate cofilin. This active form of cofilin is then able to sever and depolymerize actin filaments and thus restore the actin monomer pool. Accordingly, our data discloses the central role of Phactr4 in a feedback loop, where actin monomers regulate their own number via the activation of a key regulator of actin dynamics. Depending on the protein context, the RPEL domain can thus elicit mechanistically different responses to maintain the cellular actin balance.

Nuclear actin levels as an important transcriptional switch.

Nuclear actin levels have recently been linked to different cellular fates, suggesting that actin could act as a switch between altered transcriptional states. Here we discuss our latest results on the mechanisms by which nuclear actin levels are regulated and their implications to the functional significance of nuclear actin.

Active maintenance of nuclear actin by importin 9 supports transcription.

Besides its essential and well established role as a component of the cytoskeleton, actin is also present in the cell nucleus, where it has been linked to many processes that control gene expression. For example, nuclear actin regulates the activity of specific transcription factors, associates with all three RNA polymerases, and is a component of many chromatin remodelling complexes. Despite the fact that two export receptors, Crm1 and exportin 6, have been linked to nuclear export of actin, the mechanism by which actin enters the nucleus to elicit these essential functions has not been determined. It is also unclear whether actin is actively exchanged between the nucleus and the cytoplasm, and whether this connection has any functional significance for the cell. By applying a variety of live-cell imaging techniques we revealed that actin constantly shuttles in and out of the nucleus. The fast transport rates, which depend on the availability of actin monomers, suggest an active transport mechanism in both directions. Importantly, we identified importin 9 as the nuclear import factor for actin. Furthermore, our RNAi experiments showed that the active maintenance of nuclear actin levels by importin 9 is required for maximal transcriptional activity. Measurements of nuclear export rates and depletion studies also clarified that nuclear export of actin is mediated by exportin 6, and not by Crm1. These results demonstrate that cytoplasmic and nuclear actin pools are dynamically connected and identify the nuclear import and export mechanisms of actin.

Actin on DNA-an ancient and dynamic relationship.

In the cytoplasm of eukaryotic cells the coordinated assembly of actin filaments drives essential cell biological processes, such as cell migration. The discovery of prokaryotic actin homologues, as well as the appreciation of the existence of nuclear actin, have expanded the scope by which the actin family is utilized in different cell types. In bacteria, actin has been implicated in DNA movement tasks, while the connection with the RNA polymerase machinery appears to exist in both prokaryotes and eukaryotes. Within the nucleus, actin has further been shown to play a role in chromatin remodeling and RNA processing, possibly acting to link these to transcription, thereby facilitating the gene expression process. The molecular mechanism by which actin exerts these newly discovered functions is still unclear, because while polymer formation seems to be required in bacteria, these species lack conventional actin-binding proteins to regulate the process. Furthermore, although the nucleus contains a plethora of actin-regulating factors, the polymerization status of actin within this compartment still remains unclear. General theme, however, seems to be actin's ability to interact with numerous binding partners. A common feature to the novel modes of actin utilization is the connection between actin and DNA, and here we aim to review the recent literature to explore how this connection is exploited in different contexts.

Glycoforms of human endothelial CD34 that bind L-selectin carry sulfated sialyl Lewis x capped O- and N-glycans.

Endothelial sialomucin CD34 functions as an L-selectin ligand mediating lymphocyte extravasation only when properly glycosylated to express a sulfated carbohydrate epitope, 6-sulfo sialyl Lewis x (6-sulfo SLe(x)). It is thought that multivalent 6-sulfo SLe(x) expression promotes high-affinity binding to L-selectin by enhancing avidity. However, the reported low amount of 6-sulfo SLe(x) in total human CD34 is inconsistent with this model and prompted us to re-evaluate CD34 glycosylation. We separated CD34 into 2 glycoforms, the L-selectin-binding and nonbinding glycoforms, L-B-CD34 and L-NB-CD34, respectively, and analyzed released O- and N-glycans from both forms. L-B-CD34 is relatively minor compared with L-NB-CD34 and represented less than 10% of total tonsillar CD34. MECA-79, a mAb to sulfated core-1 O-glycans, bound exclusively to L-B-CD34 and this form contained all sulfated and fucosylated O-glycans. 6-Sulfo SLe(x) epitopes occur on core-2 and extended core-1 O-glycans with approximately 20% of total L-B-CD34 O-glycans expressing 6-sulfo SLe(x). N-glycans containing potential 6-sulfo SLe(x) epitopes were also present in L-B-CD34, but their removal did not abolish binding to L-selectin. Thus, a minor glycoform of CD34 carries relatively abundant 6-sulfo SLe(x) epitopes on O-glycans that are important for its recognition by L-selectin.

Use of bimolecular fluorescence complementation in yeast Saccharomyces cerevisiae.

Visualization of protein-protein interactions in vivo offers a powerful tool to resolve spatial and temporal aspects of cellular functions. Bimolecular fluorescence complementation (BiFC) makes use of nonfluorescent fragments of green fluorescent protein or its variants that are added as "tags" to target proteins under study. Only upon target protein interaction is a fluorescent protein complex assembled and the site of interaction can be monitored by microscopy. In this chapter, we describe the method and tools for use of BiFC in the yeast Saccharomyces cerevisiae.