Disrupting microtubule network immobilizes amoeboid chemotactic receptor in the plasma membrane.

Signaling cascades are initiated in the plasma membrane via activation of one molecule by another. The interaction depends on the mutual availability of the molecules to each other and this is determined by their localization and lateral diffusion in the cell membrane. The cytoskeleton plays a very important role in this process by enhancing or restricting the possibility of the signaling partners to meet in the plasma membrane. In this study we explored the mode of diffusion of the cAMP receptor, cAR1, in the plasma membrane of Dictyostelium discoideum cells and how this is regulated by the cytoskeleton. Single-particle tracking of fluorescently labeled cAR1 using Total Internal Reflection Microscopy showed that 70% of the cAR1 molecules were mobile. These receptors showed directed motion and we demonstrate that this is not because of tracking along the actin cytoskeleton. Instead, destabilization of the microtubules abolished cAR1 mobility in the plasma membrane and this was confirmed by Fluorescence Recovery after Photobleaching. As a result of microtubule stabilization, one of the first downstream signaling events, the jump of the PH domain of CRAC, was decreased. These results suggest a role for microtubules in cAR1 dynamics and in the ability of cAR1 molecules to interact with their signaling partners.

Single-molecule imaging of l-type Ca(2+) channels in live cells.

L-type Ca(2+) channels are an important means by which a cell regulates the Ca(2+) influx into the cytosol on electrical stimulation. Their structure and dynamics in the plasma membrane, including their molecular mobility and aggregation, is of key interest for the in-depth understanding of their function. Construction of a fluorescent variant by fusion of the yellow-fluorescent protein to the ion channel and expression in a human cell line allowed us to address its dynamic embedding in the membrane at the level of individual channels in vivo. We report on the observation of individual fluorescence-labeled human cardiac L-type Ca(2+) channels using wide-field fluorescence microscopy in living cells. Our fluorescence and electrophysiological data indicate that L-type Ca(2+) channels tend to form larger aggregates which are mobile in the plasma membrane.

Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy.

The spectral and photophysical characteristics of the autofluorescent proteins were analyzed and compared to flavinoids to test their applicability for single-molecule microscopy in live cells. We compare 1) the number of photons emitted by individual autofluorescent proteins in artificial and in vivo situations, 2) the saturation intensities of the various autofluorescent proteins, and 3) the maximal emitted photons from individual fluorophores in order to specify their use for repetitive imaging and dynamical analysis. It is found that under relevant conditions and for millisecond integration periods, the autofluorescent proteins have photon emission rates of approximately 3000 photons/ms (with the exception of DsRed), saturation intensities from 6 to 50 kW/cm2, and photobleaching yields from 10(-4) to 10(-5). Definition of a detection ratio led to the conclusion that the yellow-fluorescent protein mutant eYFP is superior compared to all the fluorescent proteins for single-molecule studies in vivo. This finding was subsequently used for demonstration of the applicability of eYFP in biophysical research. From tracking the lateral and rotational diffusion of eYFP in artificial material, and when bound to membranes of live cells, eYFP is found to dynamically track the entity to which it is anchored.

Anomalous fluorescence enhancement of Cy3 and cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin.

This study provides a critical examination of protein labeling with Cy3, Cy5, and other Cy dyes. Two alternate situations were tested. (i) Antibodies were covalently labeled with Cy dye succinimidyl ester at various fluorophore/protein ratios and the fluorescence of the labeled antibodies was compared to that of free Cy dye. (ii) Fluorescent biotin derivatives were synthesized by derivatizing ethylenediamine with one biotin and one Cy3 (or Cy5) residue. The fluorescence properties of these biotin-Cy dye conjugates were examined at all ligand/(strept)avidin ratios (0

Preparation of thiol-reactive Cy5 derivatives from commercial Cy5 succinimidyl ester.

The present study offers reliable protocols for the preparation of new thiol-reactive Cy5 derivatives which are urgently needed for single molecule fluorescence microscopy. In a systematic approach, two alternate strategies were found for the extension of commercial amine-reactive Cy5 with thiol-reactive end groups. In the two-step method, Cy5 succinimidyl ester was first reacted with ethylenediamine under conditions which gave approximately 99% asymmetric "Cy5-amine" and only approximately 1% symmetric product with two Cy5 residues. Subsequently, "Cy5-amine" was derivatized with commercial heterobifunctional cross-linkers to introduce thiol-reactive end groups (maleimide or pyridyldithio). Alternatively, commercial Cy5 succinimidyl ester was reacted with a primary amine (MTSEA, methanethiosulfonylethylamine, or PDEA, pyridyldithioethylamine) or a secondary amine (PEM, piperazinylethylmaleimide) to give the corresponding thiol-reactive derivatives in a single step. Results were good for MTSEA, moderate for PEM, and poor for PDEA. An additional drawback of the one-step method was the need for rigorous removal of unreacted Cy5 succinimidyl ester, which would label lysine residues on probe molecules. It is concluded that, except for the Cy5-MTSEA conjugate, the two-step method is much more general, reliable, and easier to follow by the typical biophysicist, biologist, etc., for whose benefit, these procedures are being published. All thiol-reactive Cy5 derivatives showed similar absorption and fluorescence properties as Cy5 succinimidyl ester, and fluorescence was fully retained after binding to thiols on proteins. The kinetics of protein labeling was also examined in order to get an idea of proper labeling conditions.

Single-molecule anisotropy imaging.

A novel method, single-molecule anisotropy imaging, has been employed to simultaneously study lateral and rotational diffusion of fluorescence-labeled lipids on supported phospholipid membranes. In a fluid membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, in which the rotational diffusion time is on the order of the excited-state lifetime of the fluorophore rhodamine, a rotational diffusion constant, D(rot) = 7 x 10(7) rad(2)/s, was determined. The lateral diffusion constant, measured by direct analysis of single-molecule trajectories, was D(lat) = 3.5 x 10(-8) cm(2)/s. As predicted from the free-volume model for diffusion, the results exhibit a significantly enhanced mobility on the nanosecond time scale. For membranes of DPPC lipids in the L(beta) gel phase, the slow rotational mobility permitted the direct observation of the rotation of individual molecules characterized by D(rot) = 1.2 rad(2)/s. The latter data were evaluated by a mean square angular displacement analysis. The technique developed here should prove itself profitable for imaging of conformational motions of individual proteins on the time scale of milliseconds to seconds.

Reorientations in the bacteriorhodopsin photoscycle are pH dependent.

Chromophore reorientations during the bacteriorhodopsin photocycle in the purple membrane of Halobacterium salinarium have been detected by time-resolved linear dichroism measurements of the optical anisotropy over the pH range from 4 to 10 and at ionic strengths from 10 mM to 1 M. The results show that reorientations in the L and M states of bacteriorhodopsin are pH dependent, reaching their largest amplitude when the membrane is at pH 6-8. Reorientations on the millisecond time scale of unexcited spectator proteins in the native purple membrane also depend on pH, consistent with the suggestion that spectator reorientations are triggered by reorientation of the photoexcited protein. The results imply that a group with a PK(a) of 5 to 6 enables reorientations, and that the deprotonation of a site at pH values above 9 restricts reorientational motion. This suggests that reorientations in M may be correlated with proton release.

Reorientations in the bacteriorhodopsin photocycle.

Reversible photoinduced reorientations of bacteriorhodopsin have been detected in suspensions of the purple membrane of Halobacterium salinarium. The anisotropy in bacteriorhodopsin during the nanosecond through millisecond stages of the photocycle was measured by time-resolved linear dichroism and transient absorption measurements. From these measurements the anisotropies of the K, L, M, and O intermediates were determined and related to the chromophore orientation with respect to the initially selected orientation. The anisotropies of the K and L states are 0.38 +/- 0.01 and 0.35 +/- 0.01, respectively. Further anisotropy decay after formation of the M intermediate in about 0.5 ms is evidence of orientational motion at this stage in the photocycle. A constant anisotropy with a value of 0.39 +/- 0.02 in the O intermediate demonstrates a recovery of the initial protein orientation with the formation of the O state. These results demonstrate that reorientations in BR are photoinduced and reversible. Similar measurements for L and M were carried out for purple membrane in polyacrylamide gels, where the anisotropies in the L and M states are 0.38 +/- 0.014 and 0.36 +/- 0.01, respectively. These results show that reorientations also occur in BR immobilized in gels. Anisotropy decay in the M state after formation of the M intermediate was not detected in the gels, in contrast to the M intermediate in suspensions. Orientational changes are observed for BR in purple membrane suspensions in the K state, during the K-->L step, in the M state possibly related to an M1-->M2 transition, and in the O state, where an almost complete return to the original orientation occurs.(ABSTRACT TRUNCATED AT 250 WORDS)