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In the past decade, extracellular vesicles (EVs) have attracted substantial interest in biomedicine. With progress in the field, we have an increasing understanding of cellular responses to EVs. In this Technical Report, we describe the direct nanoinjection of EVs into the cytoplasm of single cells of different cell lines. By using robotic fluidic force microscopy (robotic FluidFM), nanoinjection of GFP positive EVs and EV‐like particles into single live HeLa, H9c2, MDA‐MB‐231 and LCLC‐103H cells proved to be feasible. This injection platform offered the advantage of high cell selectivity and efficiency. The nanoinjected EVs were initially localized in concentrated spot‐like regions within the cytoplasm. Later, they were transported towards the periphery of the cells. Based on our proof‐of‐principle data, robotic FluidFM is suitable for targeting single living cells by EVs and may lead to information about intracellular EV cargo delivery at a single‐cell level. [ABSTRACT FROM AUTHOR]

Abstract In the past decade, extracellular vesicles (EVs) have attracted substantial interest in biomedicine. With progress in the field, we have an increasing understanding of cellular responses to EVs. In this Technical Report, we describe the direct nanoinjection of EVs into the cytoplasm of single cells of different cell lines. By using robotic fluidic force microscopy (robotic FluidFM), nanoinjection of GFP positive EVs and EV‐like particles into single live HeLa, H9c2, MDA‐MB‐231 and LCLC‐103H cells proved to be feasible. This injection platform offered the advantage of high cell selectivity and efficiency. The nanoinjected EVs were initially localized in concentrated spot‐like regions within the cytoplasm. Later, they were transported towards the periphery of the cells. Based on our proof‐of‐principle data, robotic FluidFM is suitable for targeting single living cells by EVs and may lead to information about intracellular EV cargo delivery at a single‐cell level.

The trans-Golgi network (TGN) and recycling endosome (RE) have been recognized as sorting centers, the former for newly synthesized and the latter for endocytosed proteins. However, recent findings have revealed that TGN also receives endocytosed materials and RE accepts newly synthesized proteins destined to the plasma membrane. Recently, we reported that in both Drosophila and microtubule-disrupted HeLa cells, REs are associated with the trans-side of Golgi stacks. REs are highly dynamic: their separation from and association with Golgi stacks are often observed. Importantly, a newly synthesized cargo, glycosylphosphatidylinositol-anchored-GFP was found to be concentrated in Golgi-associated REs (GA-REs), while another cargo VSVG-GFP was excluded from GA-REs before post-Golgi trafficking to the plasma membrane. This suggested that the sorting of cargos takes place at the interface of Golgi stacks and GA-REs. In this study, we demonstrated that REs could associate with Golgi stacks in sea urchin embryos, further indicating that the association of REs with Golgi stacks is a well-conserved phenomenon in the animal kingdom.

The trans-Golgi network (TGN) and recycling endosome (RE) have been recognized as sorting centers, the former for newly synthesized and the latter for endocytosed proteins. However, recent findings have revealed that TGN also receives endocytosed materials and RE accepts newly synthesized proteins destined to the plasma membrane. Recently, we reported that in both Drosophila and microtubule-disrupted HeLa cells, REs are associated with the trans-side of Golgi stacks. REs are highly dynamic: their separation from and association with Golgi stacks are often observed. Importantly, a newly synthesized cargo, glycosylphosphatidylinositol-anchored-GFP was found to be concentrated in Golgi-associated REs (GA-REs), while another cargo VSVG-GFP was excluded from GA-REs before post-Golgi trafficking to the plasma membrane. This suggested that the sorting of cargos takes place at the interface of Golgi stacks and GA-REs. In this study, we demonstrated that REs could associate with Golgi stacks in sea urchin embryos, further indicating that the association of REs with Golgi stacks is a well-conserved phenomenon in the animal kingdom. [ABSTRACT FROM AUTHOR]

The water flea Daphnia is a new model organism for ecological, evolutionary, and toxicological genomics. Detailed functional analysis of genes newly discovered through genomic approaches often requires overexpression of the identified protein. In the present study, we report the microinjection of in vitro-synthesized RNAs into the eggs as a method for overexpressing ubiquitous proteins in Daphnia magna. We injected a 1.3-kb mRNA that coded for the red fluorescent protein (DsRed2) flanked by UTRs from the ubiquitously expressed elongation factor 1α-1 ( EF1α- 1) into D. magna embryos. DsRed2 fluorescence in the embryos was measured 24 h after microinjection. Unexpectedly, the reporter RNA containing the 522-bp full-length EF1α- 1 3′ UTR failed to induce fluorescence. To assess reporter expression, the length of the 3′ UTR that potentially contained negative regulatory elements of protein expression, including AU-rich regions and Musashi binding elements, was serially reduced from the 3′ end. Assessing all injected RNA alternatives, mRNA containing the first 60 bp of the 3′ UTR gave rise to the highest fluorescence, 14 times the Daphnia auto-fluorescence. In contrast, mRNA lacking the entire 3′ UTR hardly induced any change in fluorescence intensity. This is the first evaluation of UTRs of mRNAs delivered into Daphnia embryos by microinjection for overexpressing proteins. The mRNA with truncated 3′ UTRs of Daphnia EF1α- 1 will be useful not only for gain-of-function analyses but also for labeling proteins and organelles with fluorescent proteins in Daphnia. [ABSTRACT FROM AUTHOR]

Synthetic mRNAs can be injected to achieve transient gene expression even for 'non-model' organisms in which genetic approaches are not feasible. Here, we have used this technique to express proteins that can serve as lineage tracers or reporters of cellular events in embryos of the glossiphoniid leech Helobdella robusta (phylum Annelida). As representatives of the proposed super-phylum Lophotrochozoa, glossiphoniid leeches are of interest for developmental and evolutionary comparisons. Their embryos are suitable for microinjection, but no genetic approaches are currently available. We have injected segmentation stem cells (teloblasts) with mRNAs encoding nuclear localized green fluorescent protein (nGFP) and its spectral variants, and have used tandem injections of nGFP mRNA followed by antisense morpholino oligomer (AS MO), to label single blast cell clones. These techniques permit high resolution cell lineage tracing in living embryos. We have applied them to the primary neurogenic (N) lineage, in which alternate segmental founder cells (nf and ns blast cells) contribute distinct sets of progeny to the segmental ganglia. The nf and ns blast cell clones exhibit strikingly different cell division patterns: the increase in cell number within the nf clone is roughly linear, while that in the ns clone is almost exponential. To analyze spindle dynamics in the asymmetric divisions of individual blast cells, we have injected teloblasts with mRNA encoding a tau::GFP fusion protein. Our results show that the asymmetric divisions of n blast cells result from a posterior shift of both the spindle within the cell and the midbody within the mitotic spindle, with differential regulation of these processes between nf and ns. [ABSTRACT FROM AUTHOR]

Uptake mechanisms for neutral amino acids were investigated by expression of mRNA isolated from seedlings of Ricinus communis L. in Xenopus laevis oocytes. After injection of mRNA from root, hypocotyl and cotyledon currents elicited by saccharose and neutral amino acids ranged from 0.3 nA up to 2 nA depending on the respective substrate and the source of mRNA. These currents were due to expression of low affinity uptake mechanisms and the KM values found for amino acid induced charge flow range from 1 to 2 mM. The abundance and/or the specificity of the expressed mechanisms differ in the various tissues. Currents of similar magnitude were recorded for alanine and glutamine with mRNA isolated from root, hypocotyl and cotyledons. Serine and proline induced currents after injection of mRNA from hypocotyl and roots, in case of α-aminoisobutyric acid (AIB) induced currents were generally small with mRNA from all tissues tested. In addition, differential sensitivity of glutamine and AIB uptake in the high affinity range was evident towards the amino acid analogue 2-chloro-aminophenoxybutyric acid which indicated an additional set of carriers operating in the micromolar concentration range. The results suggest that multiple transporters for neutral amino acids exist in various tissues of the plant differing in specificity of charge flow and in sensitivity towards the inhibitor 2-chloro-aminophenoxybutyric acid. [ABSTRACT FROM AUTHOR]

The epithelial Na conductance was expressed in Xenopus laevis oocytes by injection of size-fractionated mRNA of bovine tracheal epithelium. Fractionation was achieved by sucrose density gradient centrifugation. Successful expression was analysed by recording current/voltage ( I/V) curves in the presence and absence of amiloride (10 μmol/l). The newly expressed conductance was half-maximally inhibited by 44 nmol/l amiloride and exhibited a selectivity for Na over K of 140∶1. I/V curves obtained at different extracellular Na concentrations ([Na]) were subjected to a Goldman-fit analysis to obtain the relation between Na permeability ( P) and [Na]. The data show that decreasing [Na] from 85 mmol/l to 0.85 mmol/l increased P by more than threefold, which is thought to reflect Na channel inhibition by increasing [Na]. This effect clearly exceeded what can be attributed to concentration saturation of single Na channel conductance (Palmer and Frindt (1986) Proc Natl Acad Sci USA 83:2767). No correlation of inhibition with intracellular Na concentration was observed. Preservation of the [Na]-dependent self-inhibition by the newly expressed Na conductance suggests that it is an intrinsic property of the Na channel protein, probably mediated by an extracellular Na binding site. [ABSTRACT FROM AUTHOR]