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The lipid phosphatase PTEN (phosphatase and tensin homologue on chromosome 10) is a key tumour suppressor gene and an important regulator of neuronal signalling. PTEN mutations have been identified in patients with autism spectrum disorders, characterized by macrocephaly, impaired social interactions and communication, repetitive behaviour, intellectual disability, and epilepsy. PTEN enzymatic activity is regulated by a cluster of phosphorylation sites at the C-terminus of the protein. Here, we focused on the role of PTEN T366 phosphorylation and generated a knock-in mouse line in which Pten T366 was substituted with alanine (PtenT366A/T366A). We identify that phosphorylation of PTEN at T366 controls neuron size and connectivity of brain circuits involved in sensory processing. We show in behavioural tests that PtenT366/T366A mice exhibit cognitive deficits and selective sensory impairments, with significant differences in male individuals. We identify restricted cellular overgrowth of cortical neurons in PtenT366A/T366A brains, linked to increases in both dendritic arborization and soma size. In a combinatorial approach of anterograde and retrograde monosynaptic tracing using rabies virus, we characterize differences in connectivity to the primary somatosensory cortex of PtenT366A/T366A brains, with imbalances in long-range cortico-cortical input to neurons. We conclude that phosphorylation of PTEN at T366 controls neuron size and connectivity of brain circuits involved in sensory processing and propose that PTEN T366 signalling may account for a subset of autism-related functions of PTEN.

Preclinical cardiovascular research relies heavily on non-invasive in-vivo echocardiography in mice and rats to assess cardiac function and morphology, since the complex interaction of heart, circulation, and peripheral organs are challenging to mimic ex-vivo. While n-numbers of annually used laboratory animals worldwide approach 200 million, increasing efforts are made by basic scientists aiming to reduce animal numbers in cardiovascular research according to the 3R’s principle. The chicken egg is well-established as a physiological correlate and model for angiogenesis research but has barely been used to assess cardiac (patho-) physiology. Here, we tested whether the established in-ovo system of incubated chicken eggs interfaced with commercially available small animal echocardiography would be a suitable alternative test system in experimental cardiology. To this end, we defined a workflow to assess cardiac function in 8–13-day-old chicken embryos using a commercially available high resolution ultrasound system for small animals (Vevo 3100, Fujifilm Visualsonics Inc.) equipped with a high frequency probe (MX700; centre transmit: 50 MHz). We provide detailed standard operating procedures for sample preparation, image acquisition, data analysis, reference values for left and right ventricular function and dimensions, and inter-observer variabilities. Finally, we challenged incubated chicken eggs with two interventions well-known to affect cardiac physiology—metoprolol treatment and hypoxic exposure—to demonstrate the sensitivity of in-ovo echocardiography. In conclusion, in-ovo echocardiography is a feasible alternative tool for basic cardiovascular research, which can easily be implemented into the small animal research environment using existing infrastructure to replace mice and rat experiments, and thus, reduce use of laboratory animals according to the 3R principle.

The lipid phosphatase Pten (phosphatase and tensin homologue on chromosome 10) is a key tumour suppressor gene and an important regulator of neuronal signalling. Pten mutations have been identified in patients with autism spectrum disorders, characterized by macrocephaly, impaired social interactions and communication, repetitive behaviour, intellectual disability, and epilepsy. Pten enzymatic activity is regulated by a cluster of phosphorylation sites at the C-terminus of the protein. Here we specifically focussed on the role of Pten T366 phosphorylation and generated a knock-in mouse line in which Pten T366 was substituted with alanine (PtenT366A/T366A). We identify that phosphorylation of Pten at T366 controls neuron size and connectivity of brain circuits involved in sensory processing. We show in behavioural tests that PtenT366/T366A mice exhibit cognitive deficits and selective sensory impairments, with significant differences in male individuals. We identify restricted cellular overgrowth of cortical neurons in PtenT366A/T366A brains, linked to increases in both dendritic arborization and soma size. In a combinatorial approach of anterograde and retrograde monosynaptic tracing using rabies virus, we characterize differences in connectivity to the primary somatosensory cortex of PtenT366A/T366A brains, with imbalances in long-range cortico-cortical input to neurons. We conclude that phosphorylation of Pten at T366 controls neuron size and connectivity of brain circuits involved in sensory processing and propose that PTEN T366 signalling may account for a subset of autism-related functions of Pten.

Cullin 3 (Cul3) belongs to the family of cullins (Cul1–7) providing the scaffold for cullin-RING ubiquitin (Ub) ligases (CRLs), which are activated by neddylation and represent essential E3 ligases of the Ub proteasome system. During adipogenic differentiation neddylated Cul3 accumulates in LiSa-2 preadipocytes. Downregulation of Cul3 and inhibition of neddylation by MLN4924 blocks the formation of lipid droplets (LDs), the lipid storage organelles and markers of adipogenesis. Neddylation of Cul3 coincides with an increase of Rab18, a GTPase associated with LDs. Immunoprecipitation and confocal fluorescence microscopy revealed physical association of Cul3 and Rab18 at the membrane of LDs. RhoA, a suppressor of adipogenesis decreased during differentiation. Our results in LiSa-2 cells, but also mouse embryonic fibroblasts revealed a connection between Cul3, Rab18 and RhoA. Downregulation of Cul3 led to a marked increase in RhoA protein expression after 6 days of LiSa-2 cell differentiation, suggesting that Cul3 is involved in the regulation of RhoA stability.

Objective: In this study, we examined the impact of gap junction blockade on chick chorioallantoic membrane microvessels. Methods: Expression of Cx37, Cx40/42, and Cx43 in chick chorioallantoic membrane tissue was studied by PCR, Western blot, and confocal immunofluorescence microscopy. Vessel diameter changes occurring under gap junction blockade with carbenoxolone (175 ��mol/L), palmitoleic acid (100 ��mol/L), 43GAP27 (1 mmol/L) were analyzed by intravital microscopy. To analyze vascular tone, chick chorioallantoic membrane vessels were exposed to a vasodilator cocktail consisting of acetylcholine (10 ��mol/L), adenosine (100 ��mol/L), papaverine (200 ��mol/L), and sodium nitroprusside (10 ��mol/L). Results: In chick chorioallantoic membrane lysates, Western blot analysis revealed the expression of Cx40 and Cx43. Immunofluorescence in intact chick chorioallantoic membrane vasculature showed only Cx43, limited to arterial vessel walls. Upon gap junction blockade (3 hours) arterial and venous diameters decreased to 0.50 �� 0.03 and 0.36 �� 0.06 (carbenoxolone), 0.72 �� 0.08 and 0.63 �� 0.15 (palmitoleic acid) and 0.77 �� 0.004 and 0.58 �� 0.05 (GAP27), relative to initial values. Initially, diameter decrease was dominated by increasing vascular tone. After 6 hours, however, vessel tone was reduced, suggesting structural network remodeling. Conclusions: Our findings suggest a major role for connexins in mediating acute and chronic diameter changes in developing vascular networks.

The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A(2A) and A(2B) adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the gap junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of gap junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+, suppressed the 2-PAA-related enhancement of gap junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP-dependent manner. Ca2+ in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor-dependent signalling of endothelial cells of the blood-brain barrier.

Previous studies have identified alkyl-phospholipids as promising compounds for cancer therapy by targeting constituents of the cell membrane and different signaling pathways. We previously showed that the alkylphospholipid Inositol-C2-PAF inhibits the proliferation and migration of immortalized keratinocytes and the squamous carcinoma-derived cell line SCC-25. Here, we investigated the effect of this compound on growth and motility as well as its mode of action in mammary carcinoma-derived cell lines. Using BrdU incorporation and haptotactic cell migration assays, we assessed the effects of Inositol-C2-PAF on MCF-7 and MBA-MB-231 cell proliferation and migration. The phosphorylation status of signaling molecules was investigated by Western blotting as well as indirect immunofluorescence analysis and capillary isoelectric focusing. We found that Inositol-C2-PAF inhibited the growth as well as the migration in MCF-7 and MBA-MB-231 cells. Furthermore, we found that this compound inhibited phosphorylation of the protein kinase Akt at serine residue 473, but had no impact on phosphorylation at threonine 308. Phosphorylation of other kinases, such as Erk1/2, FAK and Src, which are targeted by Inositol-C2-PAF in other cells, remained unaffected by the compound in the mammary carcinoma-derived cell lines tested. In MCF-7 cells, we found that IGF-1-induced growth, as well as phosphorylation of AktS473, mTOR and the tumor suppressor pRB, was inhibited in the presence of Inositol-C2-PAF. Moreover, we found that in these cells IGF-1 had no impact on migration and did not seem to be linked to full Akt activity. Therefore, MCF-7 cell migration appears to be inhibited by Ino-C2-PAF in an Akt-independent manner. The antagonistic effects of Inositol-C2-PAF on cell migration and proliferation are indicative for its potential for breast cancer therapy, alone or in combination with other cytostatic drugs.

In most mammals, the vomeronasal system detects a variety of (semio)chemicals that mediate olfactory-driven social and sexual behaviors. Vomeronasal chemosensation depends on G protein-coupled receptors (V1R, V2R, and FPR-rs) that operate at remarkably low stimulus concentrations, thus, indicating a highly sensitive and efficient signaling pathway. We identified the PDZ domain-containing protein, Na+/H+ exchanger regulatory factor-1 (NHERF1), as putative molecular organizer of signal transduction in vomeronasal neurons. NHERF1 is a protein that contains 2 PDZ domains and a carboxy-terminal ezrin-binding domain. It localizes to microvilli of vomeronasal sensory neurons and interacts with V1Rs. Furthermore, NHERF1 and Gαi2 are closely colocalized. These findings open up new aspects of the functional organization and regulation of vomeronasal signal transduction by PDZ scaffolding proteins.

Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell-to-cell diffusion of ions, metabolites and second messengers. Stimulation of the adenosine receptor subtype AThe human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A

The scrape-loading/dye transfer technique was applied on the bovine aortic endothelial cell line GM-7373 to analyze the effects of the antithrombolytic drug dipyridamole on gap junction coupling in endothelial cells. We found that a cell treatment for 24 h with dipyridamole in therapeutically relevant concentrations (1-100 microM) increased gap junction coupling in a dose dependent manner. Similar to dipyridamole, forskolin as well as 8-Br-cAMP increased the gap junction coupling, while dibutyryl-cGMP (db-cGMP) did not affect the gap junction coupling of the GM-7373 endothelial cells. In parallel, a pharmacological inhibition of protein kinase A (PKA) with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89), antagonised the action of dipyridamole on gap junction coupling. We propose that the observed dipyridamole induced increase in gap junction coupling in endothelial cells is related to a cAMP-PKA dependent phosphorylation pathway. The report shows that gap junction coupling in endothelial cells is a suitable therapeutic target for treatment of cardiovascular diseases.

Recent results have shown that the level of gap junction coupling could modulate the induction of apoptotic reactions. We previously observed that 1H-[1,2, 4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a block- er of guanylyl cyclase, inhibited gap junction coupling and thereby promoted activation of characteristic apoptotic reactions such as chromatin condensation, DNA strand breaking, and formation of blebs in GFSHR-17 granulosa cells, the in vitro model for granulosa cells of the maturing ovular follicle. In the present report, we focus on the effects of ODQ with respect to the cell cycle in GFSHR-17 granulosa cells. In synchronised GFSHR-17 granulosa cells, the double whole-cell patch-clamp technique revealed that gap junction conductance in mitotic cells was reduced in comparison to cells in interphase. This reduction of gap junction conductance correlated with a reduction of non-phosphorylated Cx43 in mitotic cells. We compared the stimulation of apoptotic reactions by ODQ between cells in mitosis and in interphase. We observed that the induction of both chromatin condensation and DNA strand breaking by ODQ was increased in mitotic cells, as compared to cells in interphase. The effects of ODQ were not observed in He-La cells that do not express connexins. The results in- dicate that reduction of gap junction coupling in mitotic GFSHR-17 granulosa cells depends on phosphor- rylation of Cx43 and raises the sensitivity to stimulation of apoptosis. We propose that gap junction coupling is involved in regulation of apoptosis of granulosa cells in maturing ovular follicle.

Opto-perforation is an interesting alternative to conventional techniques for gene transfer into living cells. The cell membrane is perforated by femtosecond (fs) laser pulses, in order to induce an uptake of macromolecules. In this study, we successfully transfected cells with GFP vector. Transfected cells were not showing any signs of apoptosis or necrosis after 48 h. Based on simultaneously measured membrane potential changes, we calculated and experimentally verified that the relative volume exchanged is 0.4 times the total cell volume. Thus, for the first time quantitative predication of the amount of uptaken molecules and therefore a quantification of transfection is possible.

The olfactory signal transduction cascade transforms odor information into electrical signals by a cAMP-based amplification mechanism. The mechanisms underlying the very precise temporal and spatial organization of the relevant signaling components remains poorly understood. Here, we identify, using co-immunoprecipitation experiments, a macromolecular assembly of signal transduction components in mouse olfactory neurons, organized through MUPP1. Disruption of the PDZ signaling complex, through use of an inhibitory peptide, strongly impaired odor responses and changed the activation kinetics of olfactory sensory neurons. In addition, our experiments demonstrate that termination of the response is dependent on PDZ-based scaffolding. These findings provide new insights into the functional organization, and regulation, of olfactory signal transduction.

The expression and physiology of purine receptors of the human blood-brain barrier endothelial cells were characterised by application of molecular biological, gene-silencing and Ca(2+)-imaging techniques to hCMEC/D3 cells. Reverse transcription polymerase chain reaction showed the expression of the G-protein-coupled receptors P2Y(2)-, P2Y(6)-, P2Y(11)- as well as the ionotropic P2X(4)-, P2X(5)- and P2X(7)-receptors. Fura-2 ratiometry revealed that adenosine triphosphate (ATP) or uridine triphosphate (UTP) mediated a change in the intracellular Ca(2+) concentration ([Ca(2+)](i)) from 150 to 300 nM in single cells. The change in [Ca(2+)](i) corresponded to a fourfold to fivefold increase in the fluorescence intensity of Fluo-4, which was used for high-throughput experiments. Pharmacological dissection using different agonists [UTPγS, ATPγS, uridine diphosphate (UDP), adenosine diphosphate (ADP), BzATP, αβ-meATP] and antagonist (MRS2578 or NF340) as well as inhibitors of intracellular mediators (U73122 and 2-APB) showed a PLC-IP(3) cascade-mediated Ca(2+) release, indicating that the nucleotide-induced Ca(2+) signal was mainly related to P2Y(2, 6 and 11) receptors. The gene silencing of the P2Y(2) receptor reduced the ATP- or UTP-induced Ca(2+) signal and suppressed the Ca(2+) signal mediated by P2Y(6) and P2Y(11) more specific agonists like UDP (P2Y(6)), BzATP (P2Y(11)) and ATPγS (P2Y(11)). This report identifies the P2Y(2) receptor subtype as the main purine receptor involved in Ca(2+) signalling of the hCMEC/D3 cells.

The gold nanoparticle (AuNP) mediated ultrashort laser cell membrane perforation has been proven as an efficient delivery method to bring membrane impermeable molecules into the cytoplasm. Nevertheless, the underlying mechanisms have not been fully determined yet. Different effects may occur when irradiating a AuNP with ultrashort laser pulses and finally enable the molecule to transfer. Depending on the parameters (pulse length, laser fluence and wavelength, particle size and shape, etc.) light absorption or an enhanced near field scattering can lead to perforation of the cell membrane when the particle is in close vicinity. Here we present our experimental results to clarify the perforation initiating mechanisms. The generation of cavitation and gas bubbles due to the laser induced effects were observed via time resolved imaging. Additionally, pump-probe experiments for bubble detection was performed. Furthermore, in our patch clamp studies a depolarization of the membrane potential and the current through the membrane of AuNP loaded cell during laser treatment was detected. This indicates an exchange of extra- and intra cellular ions trough the perforated cell membrane for some milliseconds. Additionally investigations by ESEM imaging were applied to study the interaction of cells and AuNP after co incubation. The images show an attachment of AuNP at the cell membrane after several hours of incubation. Moreover, images of irradiated and AuNP loaded cells were taken to visualize the laser induced effects.

Whole-cell patch-clamp analysis revealed a resting membrane potential of −60 mV in primary osteoblasts and in the MG-63 osteoblast-like cells. Depolarization-induced action potentials were characterized by duration of 60 ms, a minimal peak-to-peak distance of 180 ms, a threshold value of −20 mV and a repolarization between the spikes to −45 mV. Expressed channels were characterized by application of voltage pulses between −150 mV and 90 mV in 10 mV steps, from a holding potential of −40 mV. Voltages below −60 mV induced an inward current. Depolarizing voltages above −30 mV evoked two currents: (a) a fast activated and inactivated inward current at voltages between −30 and 30 mV, and (b) a delayed-activated outward current that was induced by voltages above −30 mV. Electrophysiological and pharmacological parameters indicated that hyperpolarization activated strongly rectifying K+ (Kir) channels, whereas depolarization activated tetrodotoxin sensitive voltage gated Na+ (Nav) channels as well as delayed, slowly activated, non-inactivating, and tetraethylammonium sensitive voltage gated K+ (Kv) channels. In addition, RT-PCR showed expression of Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, and Kir2.1, Kir2.3, and Kir2.4 as well as Kv2.1. We conclude that osteoblasts express channels that allow firing of action potentials. The final publication is available at Springer via https://doi.org/10.1007/s10863-011-9354-7. BMBF/NANOTOME DFG/Transregio37 Boehringer Ingelheim International GmbH BMBF/NANOTOME DFG/Transregio/37 Boehringer Ingelheim International GmbH

The delivery of extra cellular molecules into cells is essential for cell manipulation. For this purpose genetic materials (DNA/RNA) or proteins have to overcome the impermeable cell membrane. To increase the delivery efficiency and cell viability of common methods different nano- and micro material based approaches were applied. To manipulate the cells, the membrane is in contact with the biocompatible material. Due to a field enhancement of the laser light at the material and the resulting effect the cell membrane gets perforated and extracellular molecules can diffuse into the cytoplasm. Membrane impermeable dyes, fluorescent labelled siRNA, as well as plasmid vectors encoded for GFP expression were used as an indicator for successful perforation or transfection, respectively. Dependent on the used material, perforation efficiencies over 90 % with a cell viability of about 80 % can be achieved. Additionally, we observed similar efficiencies for siRNA transfection. Due to the larger molecule size and the essential transport of the DNA into the nucleus cells are more difficult to transfect with GFP plasmid vectors. Proof of principle experiments show promising and adequate efficiencies by applying micro materials for plasmid vector transfection. For all methods a weakly focused fs laser beam is used to enable a high manipulation throughput for adherent and suspension cells. Furthermore, with these alternative optical manipulation methods it is possible to perforate the membrane of sensitive cell types such as primary and stem cells with a high viability.

The resulting effects of the interaction between nanoparticles and laser irradiation are a current matter in research. Depending on the laser parameters as well as the particles properties several effects may occur e.g. bubble formation, melting, fragmentation or an optical breakdown at the surface of the nanoparticle. Besides the investigations of these effects, we employed them to perforate the membrane of different cell lines and investigated nanoparticle mediated laser cell perforation as an alternative optical transfection method. Therefore, the gold nanoparticles (GNP) of different shapes were applied. Furthermore, we varied the methods for attaching GNP to the membrane, i.e. co-incubation of pure gold nanoparticles and bioconjugation of the surface of GNP. The optimal incubation time and the location of the GNP at the cell membrane were evaluated by multiphoton microscopy. If these GNP loaded cells are irradiated with a fs laser beam, small areas of the membrane can be perforated. Following, extra cellular molecules such as membrane impermeable dyes or foreign DNA (GFP vectors) are able to diffuse through the perforated area into the treated cells. We studied the dependence of the laser fluence, GNP concentration, GNP size and shape for successful nanoparticle mediated laser cell perforation. Due to a weak focusing of the laser beam a gentle cell treatment with high cell viabilities and high perforation efficiencies can be achieved. A further advantage of this perforation technique is the high number of cells that can be treated simultaneously. Additionally, we show applications of this method to primary and stem cells.

Purinergic signalling in rat GFSHR-17 granulosa cells was characterised by Ca(2+)-imaging and perforated patch-clamp. We observed a resting intracellular Ca(2+)-concentration ([Ca(2+)](i)) of 100 nM and a membrane potential of -40 mV. This was consistent with high K(+)- and Cl(-) permeability and a high intracellular Cl(-) concentration of 40 mM. Application of ATP for 5-15 s every 3 min induced repeated [Ca(2+)](i) increases and a 30 mV hyperpolarization. The phospholipase C inhibitor U73122 or the IP(3)-receptor antagonist 2-aminoethoethyl diphenyl borate suppressed ATP responses. Further biochemical and pharmacological experiments revealed that ATP responses were related to stimulation of P2Y(2) and P2Y(4) receptors and that the [Ca(2+)](i) increase was a prerequisite for hyperpolarization. Inhibitors of Ca(2+)-activated channels or K(+) channels did not affect the ATP-evoked responses. Conversely, inhibitors of Cl(-) channels hyperpolarized cells to -70 mV and suppressed further ATP-evoked hyperpolarization. We propose that P2Y(2) and P2Y(4) receptors in granulosa cells modulate Cl(-) permeability by regulating Ca(2+)-release.

The manipulation of cells by femtosecond (fs) laser pulses became a very important tool in cell biology. In terms of learning more about the function of the cell compartments and the cell dynamics, single cell organelles are manipulated by laser pulses. Meanwhile the cell reaction is observed by different microscopy methods. The parameters of the laser irradiation have to be chosen carefully to minimize unwanted side effects during the treatment and to prevent cell damage or cell death. In many applications, it is not known what happens due to the laser irradiation on the molecular level. The formation of reactive oxygen species (ROS) is an often predicted effect due to photo disruption in biologic tissue. In this paper, we present our study of the ROS formation during the irradiation of fs laser pulses for disruption of single cell organelles. The quantity of ROS formation depends strongly on the pulse energy of the laser. Therefore the creation of ROS was additionally studied while scanning the laser at low energy for multiphoton microscopy.

To analyse the role of PKC-dependent phosphorylation in the C-terminus of rCx46 in regulation of rCx46 connexons, truncated mutants rCx46(45.3) and rCx46(44.2) which end before and after PKC-dependent phosphorylation sites respectively were generated. Both rCx46(45.3) and rCx46(44.2) formed connexons in Xenopus oocytes similar to Cx46(wt)-connexons. They were activated by depolarisation above -40 mV and at voltages above 50 mV, inactivation was spontaneously observed or induced by PKC activator TPA, suggesting that inactivation does not require PKC-dependent phosphorylation in the C-terminus. Three casein-kinase-II-(CKII)-dependent phosphorylation sites were also identified. rCx46(37.7) and rCx46(28.2) respectively without two or all of these sites were generated. rCx46(37.7)-connexons were similar to rCx46(wt)-connexons. rCx46(28.2)-connexons comparable to rCx46(wt)-connexons were observed after injection of 50 times more rCx46(28.2)-mRNA (25 ng per oocyte). CKII-blocker inhibited depolarisation-evoked currents in oocytes injected with 0.5 ng per oocyte rCx46(37.7)-mRNA or rCx46(wt)-mRNA. Injection of 25 ng per oocyte rCx46(37.7)-mRNA or rCx46(wt)-mRNA overcame the effect of CKII-inhibitor. We propose that CKII-dependent phosphorylation in the C-terminus accelerates formation of rCx46-connexons.

Fluorescence imaging of cells and cell organelles requires labeling by fluorophores. The labeling of living cells is often done by transfection of fluorescent proteins. Viral vectors are transferring the DNA into the cell. To avoid the use of viruses, it is possible to perforate the cell membrane for example by electro-shocks, the so called electroporation, so that the fluorescent proteins can diffuse into the cell. This method causes cell death in up to 50% of the treated cells because the damage of the outer membrane is too large. A less lethal perforation of the cell membrane with high efficiency can be realized by femtosecond (fs) laser pulses. Transient pores are created by focusing the laser beam for some milliseconds on the membrane. Through this pore, the proteins can enter into the cell. This was demonstrated in a proof of principle experiment for a few cells, but it is essential to develop an opto-perforation system for large numbers of cells in order to obtain statistically significant samples for biological experiments. The relationship between pulse energy, irradiation time, repetition rate and efficacy of the transfer of a chromophor into the cells as well as the viability of the cells was analysed. The cell viability was observed up to 90 minutes after manipulation.

Fs-laser based opto-perforation is a gentle method for gene transfer into sensitive cells such as stem cells or primary cells. The high selectivity and the low damage to the cell lead to a high efficiency of transfection. However, there are side effects which induce stress to the cell due to the exchange of intra- and extracellular media as well as the disintegration of the structure of biomolecules resulting from the laser exposure. Moreover, the mechanisms of the optical transfection are still unclear. In this paper, we present our study on calcium (Ca(2+)) homeostasis during cell surgery, especially during laser induced membrane perforation. We show that the manipulation of cells can induce an increase in the cytosolic Ca(2+) concentration. This increase was not observed if the manipulation of the cells was performed in absence of the extracellular calcium indicating the importance of the Ca(2+) uptake. We found, that the uptake of extracellular Ca(2+) strongly depends on the repetition rate and the irradiation time of the laser pulses. The exposure for several seconds to kHz pulses even induces Ca(2+) induced Ca(2+) release. Dependent on the location of perforation, probably in the vicinity of an intracellular Ca(2+) stock, an instantaneous intracellular Ca(2+) release can be induced. Since Ca(2+) could be involved in negative side effect by cell surgery, we propose an application of the optoperforation technique in nominal Ca(2+)-free external solution.

Femtosecond (fs) laser-based cell surgery is typically done in two different regimes, at kHz or MHz repetition rate. Formation of reactive oxygen species (ROS) is an often predicted effect due to illumination with short laser pulses in biological tissue. We present our study on ROS formation in single cells in response to irradiation with fs laser pulses depending on the repetition rate while focusing into the cell nucleus. We observed a significant increase of ROS concentration directly after manipulation followed by a decrease in both regimes at kHz and MHz repetition rate. In addition, effects of consecutive exposures at MHz and kHz repetition rate and vice versa on ROS production were studied. Irradiation with a MHz pulse train followed by a kHz pulse train resulted in a significantly higher increase of ROS concentration than in the reversed case and often caused cell death. In the presence of the antioxidant ascorbic acid, accumulation of ROS and cell death were strongly reduced. Therefore, addition of antioxidants during fs laser-based cell surgery experiments could be advantageous in terms of suppressing photochemical damage to the cell.

Opto-perforation is an interesting alternative to conventional techniques for gene transfer into living cells. The cell membrane is perforated by femtosecond (fs) laser pulses, in order to induce an uptake of macromolecules e.g. DNA. In this study, we successfully transfected a canine cell line (MTH53a) with GFP vector or a vector coding for a GFP-HMGB1 fusion protein. The transfected cells were observed 48 hours after treatment and they were not showing any signs of apoptosis or necrosis. Based on simultaneously measured membrane potential changes during the perforation, we were able to calculate and experimentally verify that the relative volume exchanged is 0.4 times the total cell volume. Thus, for first time a quantitative predication of the amount of uptaken molecules and therefore a quantification of the transfection is possible. Additionally, this method offers new high efficient possibilities for critical transfection approaches involving special cell types, e.g. primary and stem cells.