Mattias Goksör

Uptake of analogs of penetratin, Tat(48-60) and oligoarginine in live cells.

Cell-penetrating peptides are regarded as promising vectors for intracellular delivery of large, hydrophilic molecules, but their mechanism of uptake is poorly understood. Since it has now been demonstrated that the use of cell fixation leads to artifacts in microscopy studies on the cellular uptake of such peptides, much of what has been considered as established facts must be reinvestigated using live (unfixed) cells. In this work, the uptake of analogs of penetratin, Tat(48-60), and heptaarginine in two different cell lines was studied by confocal laser scanning microscopy. For penetratin, an apparently endocytotic uptake was observed, in disagreement with previous studies on fixed cells found in the literature. Substitution of the two tryptophan residues, earlier reported to be essential for cellular uptake, did not alter the uptake characteristics. A heptaarginine peptide, with a tryptophan residue added in the C-terminus, was found to be internalized by cells via an energy-independent, non-endocytotic pathway. Finally, a crucial role for arginine residues in penetratin and Tat(48-60) was demonstrated.

Optical Manipulation Reveals Strong Attracting Forces at Membrane Contact Sites between Endoplasmic Reticulum and Chloroplasts

Eukaryote cells depend on membrane lipid trafficking from biogenic membranes, like the endoplasmic reticulum (ER), to other membranes in the cell. Two major routes for membrane lipid transport are recognized: vesicular trafficking and lipid transfer at zones of close contact between membranes. Specific ER regions involved in such membrane contact sites (MCSs) have been isolated, and lipid transfer at MCSs as well as protein-protein interactions between the partaking membranes have been demonstrated (reviewed by Holthuis, J. C. M., and Levine, T. P. (2005) Nat. Rev. 6, 209–220). Here we present the first demonstration of the physical association between membranes involved in MCSs: by using optical imaging and manipulation, strong attracting forces between ER and chloroplasts are revealed. We used Arabidopsis thaliana expressing green fluorescent protein in the ER lumen and observed leaf protoplasts by confocal microscopy. The ER network was evident, with ER branch end points apparently localized at chloroplast surfaces. After rupture of a protoplast using a laser scalpel, the cell content was released. ER fragments remained attached to the released chloroplasts and could be stretched out by optical tweezers. The applied force, 400 pN, could not drag a chloroplast free from its attached ER, which could reflect protein-protein interactions at the ER-chloroplast MCSs. As chloroplasts rely on import of ER-synthesized lipids, we propose that lipid transfer occurs at these MCSs. We suggest that lipid transfer at the MCSs also occurs in the opposite direction, for example to channel plastid-synthesized acyl groups to supply substrates for ER-localized synthesis of membrane and storage lipids.

Resonance Raman spectroscopy of optically trapped functional erythrocytes

We introduce a novel setup combining a micro-Raman spectrometer with external optical tweezers, suitable for resonance Raman studies of single functional trapped cells. The system differs from earlier setups in that two separate laser beams used for trapping and Raman excitation are combined in a double-microscope configuration. This has the advantage that the wavelength and power of the trapping and probe beam can be adjusted individually to optimize the functionality of the setup and to enable the recording of resonance Raman profiles from a single trapped cell. Trapping is achieved by tightly focusing infrared (IR) diode laser radiation (830 nm) through an inverted oil-immersion objective, and resonance Raman scattering is excited by the lines of an argon:krypton ion laser. The functionality of the system is demonstrated by measurements of trapped single functional erythrocytes using different excitation lines (488.0, 514.5, and 568.2 nm) in resonance with the heme moiety and by studying spectral evolution during illumination. We found that great care has to be taken in order to avoid photodamage caused by the visible Raman excitation, whereas the IR trapping irradiation does not seem to harm the cells or alter the hemoglobin Raman spectra. Stronger photodamage is induced by Raman excitation using 488.0- and 514.5-nm irradiation, compared with excitation with the 568.2-nm line.

Creating permanent 3D arrangements of isolated cells using holographic optical tweezers

We report the creation of permanent 3D configurations of cells, at predefined positions, within a gelatin matrix. The technique used holographic optical tweezers to manipulate individual E. coli within a solution comprising monomer precursors. The matrix was then set and after the laser beam was removed, we were able to demonstrate that the structures remained intact for many days. We were also able to demonstrate that, in the presence of appropriate nutrients, the E. coli survived within the gelatin matrix for several days. The technique could have a number of potential future applications, including the arrangement of a variety of different cell types in complex architectures, as motifs for promoting tissue differentiation and growth within the field of cell engineering.

Optical manipulation and microfluidics for studies of single cell dynamics

Most research on optical manipulation aims towards investigation and development of the system itself. In this paper we show how optical manipulation, imaging and microfluidics can be combined for investigations of single cells. Microfluidic systems have been fabricated and are used, in combination with optical tweezers, to enable environmental changes for single cells. The environment within the microfluidic system has been modelled to ensure control of the process. Three biological model systems have been studied with different combinations of optical manipulation, imaging techniques and microfluidics. In Saccharomyces cerevisiae, environmentally induced size modulations and spatial localization of proteins have been studied to elucidate various signalling pathways. In a similar manner the oxygenation cycle of single red blood cells was triggered and mapped using Raman spectroscopy. In the third experiment the forces between the endoplasmic reticulum and chloroplasts were studied in Pisum sativum and Arabidopsis thaliana. By combining different techniques we make advanced biological research possible, revealing information on a cellular level that is impossible to obtain with traditional techniques.

Sustained glycolytic oscillations in individual isolated yeast cells

Yeast glycolytic oscillations have been studied since the 1950s in cell‐free extracts and intact cells. For intact cells, sustained oscillations have so far only been observed at the population level, i.e. for synchronized cultures at high biomass concentrations. Using optical tweezers to position yeast cells in a microfluidic chamber, we were able to observe sustained oscillations in individual isolated cells. Using a detailed kinetic model for the cellular reactions, we simulated the heterogeneity in the response of the individual cells, assuming small differences in a single internal parameter. This is the first time that sustained limit‐cycle oscillations have been demonstrated in isolated yeast cells.

Osmostress-induced cell volume loss delays yeast Hog1 signaling by limiting diffusion processes and by Hog1-specific effects.

Signal transmission progresses via a series of transient protein-protein interactions and protein movements, which require diffusion within a cell packed with different molecules. Yeast Hog1, the effector protein kinase of the High Osmolarity Glycerol pathway, translocates transiently from the cytosol to the nucleus during adaptation to high external osmolarity. We followed the dynamics of osmostress-induced cell volume loss and Hog1 nuclear accumulation upon exposure of cells to different NaCl concentrations. While Hog1 nuclear accumulation peaked within five minutes following mild osmotic shock it was delayed up to six-fold under severe stress. The timing of Hog1 nuclear accumulation correlated with the degree of cell volume loss and the cells capacity to recover. Also the nuclear translocation of Msn2, the transcription factor of the general stress response pathway, is delayed upon severe osmotic stress suggesting a general phenomenon. We show by direct measurements that the general diffusion rate of Hog1 in the cytoplasm as well as its rate of nuclear transport are dramatically reduced following severe volume reduction. However, neither Hog1 phosphorylation nor Msn2 nuclear translocation were as much delayed as Hog1 nuclear translocation. Our data provide direct evidence that signaling slows down during cell volume compression, probably as a consequence of molecular crowding. Hence one purpose of osmotic adaptation is to restore optimal diffusion rates for biochemical and cell biological processes. In addition, there may be mechanisms slowing down especially Hog1 nuclear translocation under severe stress in order to prioritize Hog1 cytosolic targets.

Rapid effects of progesterone on ciliary beat frequency in the mouse fallopian tube.

BackgroundThe physiological regulation of ciliary beat frequency (CBF) within the fallopian tube is important for controlling the transport of gametes and the fertilized ovum. Progesterone influences gamete transport in the fallopian tube of several mammalian species. In fallopian tubes isolated from cows, treatment with 20 micromolar progesterone caused a rapid reduction of the tubal CBF. The aims of this study were to establish methodology for studying fallopian tube CBF in the mouse, as it is an important model species, and to investigate if progesterone rapidly affects the CBF of mice at nM concentrations.MethodsA method to assess tubal CBF of mice was developed. Fallopian tubes were dissected and the tissue was cut in small pieces. Tissue samples with moving cilia were located under an inverted bright field microscope and held still against the bottom of a petri dish by a motorized needle system. Images were acquired over 90 minutes at 35 degrees C with a high-speed camera and used for assessing changes in the CBF in response to the addition of hormone.ResultsThe baseline CBF of the mouse fallopian tube was 23.3 +/- 3.8 Hz. The CBF was stable over at least 90 minutes allowing establishment of a baseline frequency, addition of hormone and subsequent recordings. Progesterone at concentrations of 20 micromolar and 100 nM significantly reduced the CBF by 10% and 15% respectively after 30 minutes compared with controls.ConclusionsThe present study demonstrates that the mouse, despite its small size, is a useful model for studying the fallopian tube CBF ex vivo. The rapid reduction in CBF by 100 nM progesterone suggests that gamete transport in the fallopian tube could be mediated by progesterone via a non-genomic receptor mechanism.

Calibration of spatial light modulators suffering from spatially varying phase response

We present a method for converting the desired phase values of a hologram to the correct pixel addressing values of a spatial light modulator (SLM), taking into account detailed spatial variations in the phase response of the SLM. In addition to thickness variations in the liquid crystal layer of the SLM, we also show that these variations in phase response can be caused by a non-uniform electric drive scheme in the SLM or by local heating caused by the incident laser beam. We demonstrate that the use of a global look-up table (LUT), even in combination with a spatially varying scale factor, generally does not yield sufficiently accurate conversion for applications requiring highly controllable output fields, such as holographic optical trapping (HOT). We therefore propose a method where the pixel addressing values are given by a three-dimensional polynomial, with two of the variables being the (x, y)-positions of the pixels, and the third their desired phase values. The coefficients of the polynomial are determined by measuring the phase response in 8 × 8 sub-sections of the SLM surface; the degree of the polynomial is optimized so that the polynomial expression nearly replicates the measurement in the measurement points, while still showing a good interpolation behavior in between. The polynomial evaluation increases the total computation time for hologram generation by only a few percent. Compared to conventional phase conversion methods, for an SLM with varying phase response, we found that the proposed method increases the control of the trap intensities in HOT, and efficiently prevents the appearance of strong unwanted 0th order diffraction that commonly occurs in SLM systems.

Improved beam steering accuracy of a single beam with a 1D phase-only spatial light modulator

The limited number of pixels and their quantized phase modulation values limit the positioning accuracy when a phase-only one dimensional spatial light modulator (SLM) is used for beam steering. Applying the straightforward recipe for finding the optimal setting of the SLM pixels, based on individually optimizing the field contribution from each pixel to the field in the steering position, the inaccuracy can be a significant fraction of the diffraction limited spot size. This is especially true in the vicinity of certain steering angles where precise positioning is particularly difficult. However, by including in the optimization of the SLM setting an extra degree of freedom, we show that the steering accuracy can be drastically improved by a factor proportional to the number of pixels in the SLM. The extra degree of freedom is a global phase offset of all the SLM pixels which takes on a different value for each steering angle. Beam steering experiments were performed with the SLM being set both according to the conventional and the new recipe, and the results were in very good agreement with the theoretical predictions.