Fast fluorescence laser tracking microrheometry, II: quantitative studies of cytoskeletal mechanotransduction

Fluorescence laser tracking microrheometry (FLTM) is what we believe to be a novel method able to assess the local, frequency-dependent mechanical properties of living cells with nanometer spatial sensitivity at speeds up to 50 kHz. In an earlier article, we described the design, development, and optimization phases of the FLTM before reporting its performances in a variety of viscoelastic materials. In the work presented here, we demonstrate the suitability of FLTM to study local cellular rheology and obtain values for the storage and loss moduli G′(ω) and G″(ω) of fibroblasts consistent with past literature. We further establish that chemically induced cytoskeletal disruption is accompanied by reduced cellular stiffness and viscosity. Next, we provide a systematic study of some experimental variables that may critically influence microrheology measurements. First, we interrogate and justify the relevance of bead endocytosis as a method of cellular internalization of 1-μm probes in FLTM. Second, we show that as sample temperature increases, FLTM findings are elevated toward higher frequencies. Third, we confirm that relevant bead sizes (1 and 2 μm) have no effect on FLTM measurements. Fourth, we report the lack of influence of bead coatings (antiintegrin, antitransferrin, antidystroglycan, or uncoated tracers were surveyed) on their rheological readouts. Finally, we demonstrate the potential of FLTM in studying how substratum rigidity regulates cellular rheological properties. Interestingly, multiple, coupled strain relaxation mechanisms can be observed separated by two plateau moduli. Although these observations can be partly explained by rheological theories describing entangled actin filaments, there is a clear need to extend existing microrheology models to the cytoskeleton, including potentially important factors such as network geometry and remodeling.     

Chlamydomonas: Fast tracking from genomics

Elucidating biological processes has relied on the establishment of model organisms, many of which offer advantageous features such as rapid axenic growth, extensive knowledge of their physiological features and gene content, and the ease with which they can be genetically manipulated. The unicellular green alga Chlamydomonas reinhardtii has been an exemplary model that has enabled many scientific breakthroughs over the decades, especially in the fields of photosynthesis, cilia function and biogenesis, and the acclimation of photosynthetic organisms to their environment. Here, we discuss recent molecular/technological advances that have been applied to C. reinhardtii and how they have further fostered its development as a “flagship” algal system. We also explore the future promise of this alga in leveraging advances in the fields of genomics, proteomics, imaging, and synthetic biology for addressing critical future biological issues.     

High-speed, random-access fluorescence microscopy: II. Fast quantitative measurements with voltage-sensitive dyes

An improved method for making fast quantitative determinations of membrane potential with voltage-sensitive dyes is presented. This method incorporates a high-speed, random-access, laser-scanning scheme (Bullen et al., 1997. Biophys. J. 73:477-491) with simultaneous detection at two emission wavelengths. The basis of this ratiometric approach is the voltage-dependent shift in the emission spectrum of the voltage-sensitive dye di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS). Optical measurements are made at two emission wavelengths, using secondary dichroic beamsplitting and dual photodetectors (<570 nm and >570 nm). Calibration of the ratiometric measurements between signals at these wavelengths was achieved using simultaneous optical and patch-clamp measurements from adjacent points. Data demonstrating the linearity, precision, and accuracy of this technique are presented. Records obtained with this method exhibited a voltage resolution of approximately 5 mV, without any need for temporal or spatial averaging. Ratiometric recordings of action potentials from isolated hippocampal neurons are used to illustrate the usefulness of this approach. This method is unique in that it is the first to allow quantitative determination of dynamic membrane potential changes in a manner optimized for both high spatiotemporal resolution (2 micrometers and <0.5 ms) and voltage discrimination.     

Hierarchical organization of the plasma membrane: Investigations by single-molecule tracking vs. fluorescence correlation spectroscopy

Single-molecule tracking and fluorescence correlation spectroscopy (FCS) applied to the plasma membrane in living cells have allowed a number of unprecedented observations, thus fostering a new basic understanding of molecular diffusion, interaction, and signal transduction in the plasma membrane. It is becoming clear that the plasma membrane is a heterogeneous entity, containing diverse structures on nano-meso-scales (2-200 nm) with a variety of lifetimes, where certain membrane molecules stay together for limited durations. Molecular interactions occur in the time-dependent inhomogeneous two-dimensional liquid of the plasma membrane, which might be a key for plasma membrane functions.     

Delayed fluorescence from Chlorella: I. Low temperature observations

Delayed fluorescence has been observed from Chlorella whole cells at 0.5 ms following flash excitation and at temperatures from 293 °K to 120 °K. Cells which are cooled while pre-illuminated before flashes produce less observed delayed fluorescence than cells cooled without pre-illumination. There exists a small component of delayed fluorescence whose magnitude is independent of pre-illumination effects. The effect of pre-illumination upon delayed fluorescence emission is eliminated by prior freezing of the algae.     

I. Primexine development in Passiflora racemosa Brot.: overlooked aspects of development

Developmental stages during the tetrad period were examined in detail by transmission electron microscopy with an emphasis on substructure. Our purpose was to find out whether the sequence of sporoderm developmental events provides additional evidence for our recent hypothesis on the underlying cause of exine ontogeny as a sequence of self-assembling micellar mesophases initiated by genomically given physicochemical parameters. Osmiophilic globules encrusting the surface of postmeiotic microspores and tapetal cells are temporary prepattern units which come first. The second prepattern structures are highly ordered bundles of microfilaments and microtubules which determine the position of microspore surface invaginations and clusters of the glycocalyx inside them. The first glycocalyx units are microgranules which during the middle tetrad stage rearrange into radially oriented rod-like units. The latter form lens-like clusters of the glycocalyx-1, located inside the invaginations. These clusters predestine the position of the future luminae in the exine reticulum. The second glycocalyx layer is laid down as a continuous layer over the whole microspore surface and has similar substructure, that is radial rods. Glycocalyx-2 is a framework for procolumellae which appear at the late tetrad stage. Therefore, the sequence of substructural units in the primexine is: globules, microgranules, rod-like units, and layers of radially oriented rods tightly packed in the periplasmic space. This sequence corresponds to the first three mesophases of self-assembling micelles: spherical micelles, cylindrical micelles, and layers of hexagonally packed cylindrical micelles (middle mesophase). We observed the same sequence in other species during primexine development, and the findings of this study provide new evidence for our hypothesis.     

Metabolic engineering under uncertainty. I: framework development

Standard bioprocess conditions have been widely applied for the microbial conversion of raw material to essential industrial products. Successful metabolic engineering (ME) strategies require a comprehensive framework to manage the complexity embedded in cellular metabolism, to explore the impacts of bioprocess conditions on the cellular responses, and to deal with the uncertainty of the physiochemical parameters. We have recently developed a computational and statistical framework that is based on Metabolic Control Analysis and uses a Monte Carlo method to simulate the uncertainty in the values of the system parameters [Wang, L., Birol, I., Hatzimanikatis, V., 2004. Metabolic control analysis under uncertainty: framework development and case studies. Biophys. J. 87(6), 3750-3763]. In this work, we generalize this framework to incorporate the central cellular processes, such as cell growth, and different bioprocess conditions, such as different types of bioreactors. The framework provides the mathematical basis for the quantification of the interactions between intracellular metabolism and extracellular conditions, and it is readily applicable to the identification of optimal ME targets for the improvement of industrial processes [Wang, L., Hatzimanikatis, V., 2005. Metabolic engineering under uncertainty. II: analysis of yeast metabolism. Submitted].     

Picosecond laser study of fluorescence lifetimes in spinach chloroplast Photosystem I and Photosystem II preparations

Fractions enriched in either Photosystem I or Photosystem II have been prepared from chloroplasts with digitonin. A more detailed analysis of the decay kinetics of fluorescence excited by a picosecond laser pulse has been possible compared to experiments with unfractionated systems. The Photosystem I fractions show a very short component (? 100 ps) at room temperature which is apparently independent of pulse intensity over the range of photon densities used (5 · 10¹³–1 · 10¹⁶ photons cm^?2). The Photosystem II fraction has a short initial lifetime at room temperature which is strongly intensity-dependent approaching 500 ps at low photon densities, but decreasing to close to 150 ps at the highest photon densities. All of these room temperature decays appear to be non-exponential, and may possibly be fitted by at $\text{t}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ expression, expected from a random diffusion of excitations via Förster energy transfer. On cooling to 77 K, lifetimes of both Photosystem I and Photosystem II increase, the lengthening with Photosystem I being more striking. The Photosystem I decays become intensity dependent like the Photosystem II, and at the lowest photon densities decays which are more nearly exponential within the experimental error give initial lifetimes of about 2 ns. The non-exponential decays seen at high photon densities appear to fit a $\text{t}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ expression.     

Manuo-ocular coordination in target tracking. I. A model simulating human performance

During eye tracking of a self-moved target, human subjects' performance differs from eye-alone tracking of an external target. Typical latency between target and eye motion onsets is shorter, ocular smooth pursuit (SP) saturation velocity increases and the maximum target motion frequency at which the SP system functions correctly is higher. Based on a previous qualitative model, a quantitative model of the coordination control between the arm motor system and the SP system is presented and evaluated here. The model structure maintains a high level of parallelism with the physiological system. It contains three main parts: the eye motor control (containing a SP branch and a saccadic branch), the arm motor control and the coordination control. The coordination control is achieved via an exchange of information between the arm and the eye sensorimotor systems, mediated by sensory signals (vision, proprioception) and motor command copy. This cross-talk results in improved SP system performance. The model has been computer simulated and the results have been compared with human subjects' behavior observed during previous experiments. The model performance is seen to quantitatively fit data on human subjects. Received: 6 March 1997 / Accepted in revised form: 15 July 1997     

PMS: Photosystem I electron donor or fluorescence quencher

Light energy harvested by the pigments in Photosystem I (PSI) is used for charge separation in the reaction center (RC), after which the positive charge resides on a special chlorophyll dimer called P700. In studies on the PSI trapping kinetics, P700(+) is usually chemically reduced to re-open the RCs. So far, the information available about the reduction rate and possible chlorophyll fluorescence quenching effects of these reducing agents is limited. This information is indispensible to estimate the fraction of open RCs under known experimental conditions. Moreover, it would be important to understand if these reagents have a chlorophyll fluorescence quenching effects to avoid the introduction of exogenous singlet excitation quenching in the measurements. In this study, we investigated the effect of the commonly used reducing agent phenazine methosulfate (PMS) on the RC and fluorescence emission of higher plant PSI-LHCI. We measured the P700(+) reduction rate for different PMS concentrations, and show that we can give a reliable estimation on the fraction of closed RCs based on these rates. The data show that PMS is quenching chlorophyll fluorescence emission. Finally, we determined that the fluorescence quantum yield of PSI with closed RCs is 4% higher than if the RCs are open.     

Cyanobacterial picoplankton from Lake Constance. I. Isolation by fluorescence characteristics

Unicellular autotrophs ranging from 0 9 to 4 (jun in diameterwere isolated from Lake Constance by plating aliquots of wateron solidified mineral medium (BG11) Single colonies were selectedfor further cultivation using epifluorescence microscopy andfluorescence emission spectroscopy to identify species differingin pigment composition The cyanobacterial isolates representthree pigment types containing phycobilisomes with either phycocyaninor phycoerythnn as dominant accessory pigment or a newly described,highly fluorescent phycocyanin-like pigment The differencesfound in fluorescence emission of isolated clones are discussedwith respect to in situ strain identification Present address: Michigan State University, MSU-DOE Plant ResearchLaboratory, East Lansing, MI 48824-1312, USA     

Intramolecular electron transfer in diastereomeric naphthalene-amine dyads: a fluorescence and laser flash photolysis study.

Two dyads containing a naphthalene-like chromophore linked to a pyrrolidine-derived moiety, namely (S,S)- and (R,S)-NPX-PYR, have been synthesised by esterification of (S)- or (R)-naproxen (NPX) with (S)-N-methyl-2-pyrrolidinemethanol (PYR) and submitted to photophysical studies (steady-state and time-resolved fluorescence, as well as laser flash photolysis). The emission spectra of the dyads in acetonitrile were characterised by a typical band centred at 350 nm, identical to that of the reference compound (S)-NPX. However the intensities were clearly different, revealing a significant intramolecular quenching in the dyads, as well as a remarkable stereodifferentiation (factor of 1.6). Accordingly, the fluorescence lifetimes of the two dyads were different from each other and markedly shorter than that of (S)-NPX. The quenching mechanism is intramolecular electron transfer, that is thermodynamically favoured. Exciplex formation, that is nearly thermoneutral, does not compete efficiently. The electron transfer rate constants for (S,S)- and (R,S)-(NPX-PYR) were 1.8 x 10(8) and 2.8 x 10(8) s(-1), respectively. By contrast, no significant intramolecular quenching was observed for the excited triplet states (lambda(max)= 440 nm), generated by laser flash photolysis; this is in agreement with the fact that intramolecular electron transfer is thermodynamically disfavoured, due to the lower energy of excited triplets.     

Bovine inositol monophosphatase: development of a continuous fluorescence assay of enzyme activity.

This paper describes a continuous assay for the enzyme inositol monophosphatase which has been developed using a new substrate, the fluorescent compound 4-methylumbelliferyl phosphate. The hydrolysis of the phosphate group from this compound can be readily detected by a resultant large red shift in the emission spectrum from 390-450 nm. The kinetic constants for the enzyme using this new substrate are described.     

Static and dynamic quenching of protein fluorescence. I. Bovine serum albumin.

The fluorescence parameters, lifetime, relative quantum yield, maximum and mean wavelength, half-width, and polarization, of bovine serum albumin (BSA) were measured at 15°C in aqueous solutions containing varying concentrations of different chemical perturbants, glycerol, Cu²⁺ ions, guanidine hydrochloride, and urea. By considering a quenching mechanism as being either dynamic or static, depending upon whether the quenching is or is not accompanied by a change in the fluorescence lifetime, we were able to correlate the changes produced in the various fluorescence parameters by the different chemical perturbants with changes in macromolecular structure as the concentration of perturbant was gradually increased. The addition of glycerol and of Cu²⁺ ions indicated that in aqueous BSA both tryptophan residues are below the surface of the macromolecule, out of contact with solvent water, and, as a consequence, they are statically quenched. “Ultra-Pure” guanidine hydrochloride at 2.4 M or more caused a drastic conformation change, which resulted in the emergence of a visible tyrosine peak at 304 nm in the BSA fluorescence spectrum when either 260- or 270-nm excitation was employed. With the same excitation, the enhancement of BSA tyrosine fluorescence by 6–8 M ultra-pure urea produced only a shoulder near 304 nm in the BSA fluorescence spectrum. We have introduced the use of a new relative quantum yield for protein fluorescence, q′, referenced to the quantum yield of unquenched free tryptophan, which eliminates the quenching action of water from the reference.     

Scanning fluorescence correlation spectroscopy. I. Theory and simulation of aggregation measurements.

Scanning Fluorescence Correlation Spectroscopy (S-FCS) is introduced as an adaptation of Fluorescence Correlation Spectroscopy (FCS) to measure aggregation in systems, such as biological cell membranes, where diffusion or flow is slow. The theoretical framework for interpretation of S-FCS measurements are discussed in this paper with emphasis on the limitations arising from the sample size and shape. Computer simulations of the experiment demonstrate the potential of the technique and illustrate how some of the limitations may be overcome.