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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 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.

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.

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 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.

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.

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.

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.