Determination of diffusion coefficient by image-based fluorescence recovery after photobleaching and single particle tracking system implemented in a single platform

Fluorescence recovery after photobleaching(FRAP)and single particle tracking(SPT)techni-ques determine the diffusion coefficient from average diffusive motion of high-concentration molecules and from trajectories of low-concentration single molecules,respectively.Lateral dif-fusion coefficients measured by FRAP and SPT techniques for the same biomolecule on cell membrane have exhibited inconsistent values across laboratories and platforms with larger dif-fusion coefficient determined by FRAP,but the sources of the inconsistency have not been investigated thoroughly.Here,we designed an image-based FRAP-SPT system and made a direct comparison between FRAP and SPT for diffusion coefficient of submicron particles with known theoretical values derived from Stokes-Einstein equation in aqueous solution.The combined iFRAP-SPT technique allowed us to measure the diffusion coefficient of the same fluorescent particle by utilizing both techniques in a single platform and to scrutinize inherent errors and artifacts of FRAP.Our results reveal that diffusion coefficient overestimated by FRAP is caused by inaccurate estimation of the bleaching spot size and can be corrected by simple image analysis.Our iFRAP-SPT technique can be potentially used for not only cellular membrane dynamics but also for quantitative analysis of the spatiotemporal distribution of the solutes in small scale analytical devices.

Fluorescence recovery after photobleaching:analyses of cyanobacterial phycobilisomes reveal intrinsic fluorescence recovery

Fluorescence recovery after photobleaching(FRAP)has been used to study the dynamics of the cyanobacterial photosynthesis apparatus since 1997.Fluorescence recovery of cyanobacteria during FRAP was conventionally interpreted as a result of phycobilisome(PBS)diffusion on the surface of the thylakoid membrane.The mechanism of state transition in cyanobacteria has been widely attributed to PBS diffusion.However,in red algae,another PBS-containing group,the intrinsic photoprocess was found to contribute greatly to the fluorescence recovery of PBS,which raises questions concerning the role of FRAP in red algal PBS.Therefore,it is important to re-evaluate the nature of PBS fluorescence recovery in cyanobacteria.In the present study,four cyanobacterial strains with different phenotypes and PBS compositions were used to investigate their FRAP characteristics.Fluorescence recovery of PBS was observed in wholly photobleached cells in all four cyanobacterial strains,in which the contribution of PBS diffusion to the fluorescence recovery was not possible.Moreover,the fluorescence recovered in isolated PBSs and PBS-thylakoid membranes after photobleaching further demonstrated the intrinsic photoprocess nature of fluorescence recovery.These findings suggest that the intrinsic photoprocess contributed to the fluorescence recovery following photobleaching when measured by the FRAP method.

Recovery of PET-quenched Fluorescent Conjugated Polymers with Aza-15-crown-5 as Pendant Groups on Interaction with Ions

Novel conjugated polymers with aza-15-crown-5 as pendant groups, P1 were synthesized by the Sonogashira coupling reaction. The polymer P1 was found to show fluorescence en-hancement response towards Ba2＋. The gradual recovery and turning-point of emission intensity of polymers appeared in the addition of Ba2＋, which provided good evidence for the fluorescence amplification mechanism of conjugated polymers.

Laser Stimulation of the Chloroplast/Endoplasmic Reticulum Nexus in Tobacco Transiently Produces Protein Aggregates （Boluses） within the Endoplasmic Reticulum and Stimulates Local ER Remodeling

Does the ER subdomain that associates with the chloroplast in vivo, hereafter referred to as the chloroplast/ER nexus, play a role in protein flow within the ER？ In studies of tobacco cells either constitutively or transiently expressing ER-retained luminal, GFP-HDEL, or trans-membrane, YFP-RHD3, fluorescent fusion proteins, brief 405-nm （3-6-mW） laser stimulation of the nexus causes a qualitative difference in the movement and behavior of proteins in the ER. Photostimulating the nexus produces fluorescent protein punctate aggregates （boluses） within the lumen and membrane of the ER. The aggregation propagates through the membrane network throughout the cell, but within minutes can revert to normal, with disaggregation propagating back toward the originally photostimulated nexus. In the meantime, the ER grows and anastomoses around the chloroplast, forming a dense cisternal and tubular network. If this network is again photostimulated, bolus formation does not recur and, if the photostimulation results in photobleaching, fluorescence recovery after photobleaching occurs as it would typically in areas away from the nexus. Bolus propagation is not mediated by the actin cytoskeleton, but can be reversed by pre-conditioning the cells for 30 min with high, 40-45℃, temperature （heat stress）. Because it is not reversed with heat stress, the reorganization of the ER at the nexus following photostimulation is a separate event.

HID-1 is a peripheral membrane protein primarily associated with the medial-and transGolgi apparatus

Caenorhabditis elegans hid-1 gene was first identified in a screen for mutants with a high-temperature-induced dauer formation(Hid)phenotype.Despite the fact that the hid-1 gene encodes a novel protein(HID-1)which is highly conserved from Caenorhabditis elegans to mammals,the domain structure,subcellular localization,and exact function of HID-1 remain unknown.Previous studies and various bioinformatic softwares predicted that HID-1 contained many transmembrane domains but no known functional domain.In this study,we revealed that mammalian HID-1 localized to the medial-and transGolgi apparatus as well as the cytosol,and the localization was sensitive to brefeldin A treatment.Next,we demonstrated that HID-1 was a peripheral membrane protein and dynamically shuttled between the Golgi apparatus and the cytosol.Finally,we verified that a conserved N-terminal myristoylation site was required for HID-1 binding to the Golgi apparatus.We propose that HID-1 is probably involved in the intracellular trafficking within the Golgi region.