Fluorescence Loss In Photobleaching Research Articles

The Alphabet Soup of Microscopy: An Introduction to Advanced Imaging Techniques. Part II: What the FLIP, FLAP, FRAP, FRET, and FLIM is Going On?

Abstract The use of fluorescence in imaging has been a pivotal factor in advancing our scientific understanding. As microscopy continues to evolve, the terminology used to describe these techniques becomes increasingly complex, often resulting in a bewildering array of acronyms that resemble alphabet soup. Among the most prominent acronyms are those associated with advanced fluorescence microscopy techniques: Fluorescence Loss in Photobleaching (FLIP), Fluorescence Localization after Photobleaching (FLAP), Fluorescence Recovery after Photobleaching (FRAP), Förster Resonance Energy Transfer (FRET), and Fluorescence Lifetime Imaging Microscopy (FLIM). Each of these methods provides invaluable insights into molecular dynamics and interactions within living cells and tissues. This article, Part 2 of the Alphabet Soup of Microscopy series, aims to clarify these widely used fluorescence microscopy techniques and to illuminate their contributions to our understanding of cellular processes.

Evidence of the predominance of passive symplastic phloem loading and sugar transport with leaf ageing in Camellia oleifera

Phloem loading and transport of sugar from leaves to sink tissues such as fruits are crucial for yield formation. Camellia oleifera is an evergreen horticultural crop with high value; however, its low production limits the development of the C. oleifera industry. In this study, using a combination of ultrastructural observation, fluorescence loss in photobleaching (FLIP) and inhibitor treatment, we revealed that C. oleifera leaves mainly adopt a symplastic loading route from mesophyll cells to the surrounding vascular bundle cells in minor veins. HPLC assays showed that sucrose is the main sugar transported and only a small amount of raffinose or stachyose was detected in petioles, supporting a passive symplastic loading route in C. oleifera leaves. Compared to leaves grown this year (LT), the carbohydrate synthesis capacity in leaves grown last year (LL) was decreased while LL retained more soluble sugar, suggesting a decrease in transport capacity with leaf ageing. TEM and tissue staining showed that a reduction in plasmodesmata density leads to a decline in the degree of cellular coupling and is responsible for the weakening transport capacity in older leaves. RNA-seq revealed several differentially expressed genes (DEGs) including CoPDCB1-1, CoSUT1 and CoSWEET12, which are likely involved in the regulation of phloem loading and sugar transport. An expression correlation network is constructed between PD-callose binding protein genes, sugar transporter genes and senescence-associated genes. Collectively, this study provides the evidence of the passive symplastic phloem loading pathway in C. oleifera leaves and constructs the correlation between sugar transport and leaf ageing.

Bad is essential for Bcl-xL-enhanced Bax shuttling between mitochondria and cytosol.

Although Bcl-xL has been shown to retrotranslocate Bax from mitochondria to cytosol, other studies have found that Bcl-xL also stabilizes the mitochondrial localization of Bax. It is still unclear what causes the difference in Bcl-xL-regulated Bax localization. Bad, a BH3-only protein with a high affinity for Bcl-xL, may play an important role in Bcl-xL-regulated Bax shuttling. Here, we found that Bcl-xL enhanced both translocalization and retrotranslocation of mitochondrial Bax, as evidenced by quantitative co-localization, western blots and fluorescence loss in photobleaching (FLIP) analyses. Notably, Bad knockdown prevented Bcl-xL-mediated Bax retrotranslocation, indicating Bad was essential for this process. Quantitative fluorescence resonance energy transfer (FRET) imaging in living cells and co-immunoprecipitation analyses showed that the interaction of Bcl-xL with Bad was stronger than that with Bax. The Bad mimetic ABT-737 dissociated Bax from Bcl-xL on isolated mitochondria, suggesting that mitochondrial Bax was directly liberated to cytosol due to Bad binding to Bcl-xL. In addition, MK-2206, an Akt inhibitor, decreased Bad phosphorylation while increasing cytosolic Bax proportion. Our data firmly demonstrate a notion that Bad binds to mitochondrial Bcl-xL to release Bax, resulting in retrotranslocation of Bax to cytosol, and that the amount of Bad involved is regulated by Akt signaling.

Statin-induced increase in actin polymerization modulates GPCR dynamics and compartmentalization

The function of the actin cytoskeleton in cellular motility and trafficking has been widely studied. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol and its consequences on membrane dynamics are addressed only rarely. In a recent work, we reported that chronic cholesterol depletion using statins leads to significant polymerization of the actin cytoskeleton. In this work, we explore the effect of reorganization of the actin cytoskeleton on membrane dynamics under cholesterol-depleted condition. Specifically, we explore the role of actin cytoskeleton in regulating the dynamics of the serotonin1A receptor, a crucial neurotransmitter G protein-coupled receptor (GPCR) that plays a major role in the generation and modulation of cognitive and behavioral functions. For this, we analyzed the lateral dynamics of the serotonin1A receptor in cholesterol-depleted cells (using statins) by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) measurements. Our results indicate that lateral diffusion parameters of serotonin1A receptors in normal cells are consistent with models describing the diffusion of molecules in a homogeneous membrane. Interestingly, these parameters are altered in cholesterol-depleted cells and the receptor exhibits dynamic confinement. Notably, our results show that statin-induced dynamic confinement could be reversed by destabilization of the actin cytoskeleton. On a broader perspective, these results assume significance in understanding the modulatory role of the membrane environment on the organization and dynamics of GPCRs in diseases caused by altered cholesterol biosynthesis.

Human neural progenitors establish a diffusion barrier in the endoplasmic reticulum membrane during cell division.

ABSTRACTAsymmetric segregation of cellular components regulates the fate and behavior of somatic stem cells. Similar to dividing budding yeast and precursor cells in Caenorhabditis elegans, it has been shown that mouse neural progenitors establish a diffusion barrier in the membrane of the endoplasmic reticulum (ER), which has been associated with asymmetric partitioning of damaged proteins and cellular age. However, the existence of an ER diffusion barrier in human cells remains unknown. Here, we used fluorescence loss in photobleaching (FLIP) imaging to show that human embryonic stem cell (hESC)- and induced pluripotent stem cell (iPSC)-derived neural progenitor cells establish an ER diffusion barrier during cell division. The human ER diffusion barrier is regulated via lamin-dependent mechanisms and is associated with asymmetric segregation of mono- and polyubiquitylated damaged proteins. Further, forebrain regionalized organoids derived from hESCs were used to show the establishment of an ER membrane diffusion barrier in more naturalistic tissues, mimicking early steps of human brain development. Thus, the data provided here show that human neural progenitors establish a diffusion barrier during cell division in the membrane of the ER, which may allow for asymmetric segregation of cellular components, contributing to the fate and behavior of human neural progenitor cells.

Dynamic Mode Decomposition of Fluorescence Loss in Photobleaching Microscopy Data for Model-Free Analysis of Protein Transport and Aggregation in Living Cells.

The phase separation and aggregation of proteins are hallmarks of many neurodegenerative diseases. These processes can be studied in living cells using fluorescent protein constructs and quantitative live-cell imaging techniques, such as fluorescence recovery after photobleaching (FRAP) or the related fluorescence loss in photobleaching (FLIP). While the acquisition of FLIP images is straightforward on most commercial confocal microscope systems, the analysis and computational modeling of such data is challenging. Here, a novel model-free method is presented, which resolves complex spatiotemporal fluorescence-loss kinetics based on dynamic-mode decomposition (DMD) of FLIP live-cell image sequences. It is shown that the DMD of synthetic and experimental FLIP image series (DMD-FLIP) allows for the unequivocal discrimination of subcellular compartments, such as nuclei, cytoplasm, and protein condensates based on their differing transport and therefore fluorescence loss kinetics. By decomposing fluorescence-loss kinetics into distinct dynamic modes, DMD-FLIP will enable researchers to study protein dynamics at each time scale individually. Furthermore, it is shown that DMD-FLIP is very efficient in denoising confocal time series data. Thus, DMD-FLIP is an easy-to-use method for the model-free detection of barriers to protein diffusion, of phase-separated protein assemblies, and of insoluble protein aggregates. It should, therefore, find wide application in the analysis of protein transport and aggregation, in particular in relation to neurodegenerative diseases and the formation of protein condensates in living cells.

Electrochemiluminescence Loss in Photobleaching

AbstractThe effects of photobleaching on electrochemiluminescence (ECL) was investigated for the first time. The plasma membrane of Chinese Hamster Ovary (CHO) cells was labeled with a [Ru(bpy)3]2+ derivative. Selected regions of the fixed cells were photobleached using the confocal mode with sequential stepwise illumination or cumulatively and they were imaged by both ECL and photoluminescence (PL). ECL was generated with a model sacrificial coreactant, tri‐n‐propylamine. ECL microscopy of the photobleached regions shows lower ECL emission. We demonstrate a linear correlation between the ECL decrease and the PL loss due to the photobleaching of the labels immobilized on the CHO membranes. The presented strategy provides valuable information on the fundamentals of the ECL excited state and opens new opportunities for exploring cellular membranes by combining ECL microscopy with photobleaching techniques such as fluorescence recovery after photobleaching (FRAP) or fluorescence loss in photobleaching (FLIP) methods.

Niemann Pick C2 protein enables cholesterol transfer from endo-lysosomes to the plasma membrane for efflux by shedding of extracellular vesicles

The Niemann-Pick C2 protein (NPC2) is a sterol transfer protein in the lumen of late endosomes and lysosomes (LE/LYSs). Absence of functional NPC2 leads to endo-lysosomal buildup of cholesterol and other lipids. How NPC2’s known capacity to transport cholesterol between model membranes is linked to its function in living cells is not known. Using quantitative live-cell imaging combined with modeling of the efflux kinetics, we show that NPC2-deficient human fibroblasts can export the cholesterol analog dehydroergosterol (DHE) from LE/LYSs. Internalized NPC2 accelerated sterol efflux extensively, accompanied by reallocation of LE/LYSs containing fluorescent NPC2 and DHE to the cell periphery. Using quantitative fluorescence loss in photobleaching of TopFluor-cholesterol (TF-Chol), we estimate a residence time for a rapidly exchanging sterol pool in LE/LYSs localized in close proximity to the plasma membrane (PM), of less than one min and observed non-vesicular sterol exchange between LE/LYSs and the PM. Excess sterol was released from the PM by shedding of cholesterol-rich vesicles. The ultrastructure of such vesicles was analyzed by combined fluorescence and cryo soft X-ray tomography (SXT), revealing that they can contain lysosomal cargo and intraluminal vesicles. Treating cells with apoprotein A1 and with nuclear receptor liver X-receptor (LXR) agonists to upregulate expression of ABC transporters enhanced cholesterol efflux from the PM, at least partly by accelerating vesicle release. We conclude that NPC2 inside LE/LYSs facilitates non-vesicular sterol exchange with the PM for subsequent sterol efflux to acceptor proteins and for shedding of sterol-rich vesicles from the cell surface.

RanBP1 controls the Ran pathway in mammalian cells through regulation of mitotic RCC1 dynamics

ABSTRACT The Ran GTPase plays critical roles in multiple cellular processes including interphase nucleocytoplasmic transport and mitotic spindle assembly. During mitosis in mammalian cells, GTP-bound Ran (Ran-GTP) is concentrated near mitotic chromatin while GDP-bound Ran (Ran-GDP) is more abundant distal to chromosomes. This pattern spatially controls spindle formation because Ran-GTP locally releases spindle assembly factors (SAFs), such as Hepatoma Up-Regulated Protein (HURP), from inhibitory interactions near chromosomes. Regulator of Chromatin Condensation 1 (RCC1) is Ran’s chromatin-bound exchange factor, and RanBP1 is a conserved Ran-GTP-binding protein that has been implicated as a mitotic regulator of RCC1 in embryonic systems. Here, we show that RanBP1 controls mitotic RCC1 dynamics in human somatic tissue culture cells. In addition, we observed the re-localization of HURP in metaphase cells after RanBP1 degradation, consistent with the idea that altered RCC1 dynamics functionally modulate SAF activities. Together, our findings reveal an important mitotic role for RanBP1 in human somatic cells, controlling the spatial distribution and magnitude of mitotic Ran-GTP production and thereby ensuring the accurate execution of Ran-dependent mitotic events. Abbreviations AID: Auxin-induced degron; FLIP: Fluorescence loss in photobleaching; FRAP: Fluorescence recovery after photobleaching; GDP: guanosine diphosphate; GTP: guanosine triphosphate; HURP: Hepatoma Up-Regulated Protein; NE: nuclear envelope; NEBD: Nuclear Envelope Breakdown; RanBP1: Ran-binding protein 1; RanGAP1: Ran GTPase-Activating Protein 1; RCC1: Regulator of Chromatin Condensation 1; RRR complex: RCC1/Ran/RanBP1 heterotrimeric complex; SAF: Spindle Assembly Factor; TIR1: Transport Inhibitor Response 1 protein; XEE: Xenopus egg extract.

Anti-apoptotic capacity of Mcl-1Δ127

The anti-apoptotic ability of Mcl-1Δ127, a caspase cleavage product of Mcl-1, is debated. We here used fluorescence imaging to assess the anti-apoptotic capacity of Mcl-1Δ127 in living cells. Fluorescence imaging of living cells expressing CFP-Mcl-1Δ127 showed that Mcl-1Δ127 existed mainly in cytoplasm. Fluorescence imaging of living cells co-expressing CFP-Mcl-1Δ127 and YFP-Bak, CFP-Mcl-1Δ127 and YFP-BimL, CFP-Mcl-1Δ127 and YFP-Puma or CFP-Mcl-1Δ127 and YFP-tBid showed that Mcl-1Δ127 markedly inhibited the oligomerization of Bak, BimL, Puma and tBid on mitochondria and also inhibited the Bak-, BimL-, Puma- or tBid-mediated cell death, resulting in their partial localization in cytoplasm. Fluorescence resonance energy transfer (FRET) imaging proved that Mcl-1Δ127 bound to Bak, BimL, Puma and tBid, respectively. Fluorescence loss in photobleaching (FLIP) analyses showed that Mcl-1Δ127 did prevent Bak oligomerization by retrotranslocating Bak from mitochondria into cytoplasm. Collectively, Mcl-1Δ127 has the same anti-apoptotic capacity as Mcl-1, and prevents apoptosis by sequestering BH3-only or Bak proteins, thus inhibiting their oligomerization on mitochondria.

Bcl-xL inhibits PINK1/Parkin-dependent mitophagy by preventing mitochondrial Parkin accumulation.

This report aims to explore how Bcl-xL, a Bcl-2 family protein, regulates PINK1/Parkin-dependent mitophagy. Compared with the Hela cells expressing Parkin alone, co-expression of Bcl-xL significantly inhibited CCCP (Carbonyl cyanide 3- chlorophenylhydrazone)-induced mitochondrial Parkin accumulation and mitophagy. Western blotting analysis illustrated that over-expressed Bcl-xL inhibited CCCP-induced decrease of mitochondrial proteins in Parkin over-expressed cells. Fluorescence loss in photobleaching (FLIP) analyses demonstrated that Bcl-xL inhibited the CCCP-induced translocation of Parkin into mitochondria not by retrotranslocating Parkin from mitochondria to cytoplasm. Fluorescence resonance energy transfer (FRET) imaging revealed in Hela cells that Bcl-xL physically bound with Parkin to form oligomer in cytoplasm, and that Bcl-xL also directly interacted with PINK1 on mitochondria. analysis for HEK293 T cells verified that endogenous Bcl-xL interacted with both endogenous Parkin and PINK1. Collectively, Bcl-xL inhibits PINK1/Parkin- dependent mitophagy by preventing the accumulation of Parkin on mitochondria via two regulation ways: directly binds to Parkin in cytoplasm to prevent the translocation of Parkin from cytoplasm to mitochondria and directly binds to PINK1 on mitochondria to inhibit the Parkin from cytoplasm to mitochondria by PINK1.

Atg38-Atg8 interaction in fission yeast establishes a positive feedback loop to promote autophagy

ABSTRACT Macroautophagy (autophagy) is driven by the coordinated actions of core autophagy-related (Atg) proteins. Atg8, the core Atg protein generally considered acting most downstream, has recently been shown to interact with other core Atg proteins via their Atg8-family-interacting motifs (AIMs). However, the extent, functional consequence, and evolutionary conservation of such interactions remain inadequately understood. Here, we show that, in the fission yeast Schizosaccharomyces pombe, Atg38, a subunit of the phosphatidylinositol 3-kinase (PtdIns3K) complex I, interacts with Atg8 via an AIM, which is highly conserved in Atg38 proteins of fission yeast species, but not conserved in Atg38 proteins of other species. This interaction recruits Atg38 to Atg8 on the phagophore assembly site (PAS) and consequently enhances PAS accumulation of the PtdIns3K complex I and Atg proteins acting downstream of the PtdIns3K complex I, including Atg8. The disruption of the Atg38-Atg8 interaction leads to the reduction of autophagosome size and autophagic flux. Remarkably, the loss of this interaction can be compensated by an artificial Atg14-Atg8 interaction. Our findings demonstrate that the Atg38-Atg8 interaction in fission yeast establishes a positive feedback loop between Atg8 and the PtdIns3K complex I to promote efficient autophagosome formation, underscore the prevalence and diversity of AIM-mediated connections within the autophagic machinery, and reveal unforeseen flexibility of such connections. Abbreviations: AIM: Atg8-family-interacting motif; AP-MS: affinity purification coupled with mass spectrometry; Atg: autophagy-related; FLIP: fluorescence loss in photobleaching; PAS: phagophore assembly site; PB: piggyBac; PE: phosphatidylethanolamine; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate.

BCL-XL directly retrotranslocates the monomeric BAK

BCL-XL, an anti-apoptotic BCL-2 family protein, potently inhibits BAK oligomerization and the formation of toxic mitochondrial pores in response to cellular stress. This report aims to explore which form of mitochondrial monomeric and oligomerized BAK can be retrotranslocated by BCL-XL. Fluorescence imaging of living cells co-expressing CFP-BCL-XL and YFP-BAK showed that BCL-XL markedly inhibited mitochondrial BAK oligomerization and resulted in partial cytosolic BAK distribution. Live-cell fluorescence resonance energy transfer (FRET) analyses showed that BAK auto-oligomerized on mitochondria and BCL-XL physically sequestrated monomeric BAK to prevent BAK oligomerization. Fluorescence loss in photobleaching (FLIP) analyses showed that BCL-XL retrotranslocated the monomeric BAK from mitochondria into cytosol, whereas monomeric BAK reduced the retrotranslocation rate of BCL-XL. Live-cell time-lapse imaging and FLIP experiments in living cells with BAK oligomers displayed that BCL-XL did not depolymerize or retrotranslocate the oligomerized BAK. Collectively, BCL-XL retrotranslocates monomeric instead of oligomerized BAK from mitochondria into cytosol.

A discontinuous Galerkin model for fluorescence loss in photobleaching of intracellular polyglutamine protein aggregates

BackgroundIntracellular phase separation and aggregation of proteins with extended poly-glutamine (polyQ) stretches are hallmarks of various age-associated neurodegenerative diseases. Progress in our understanding of such processes heavily relies on quantitative fluorescence imaging of suitably tagged proteins. Fluorescence loss in photobleaching (FLIP) is particularly well-suited to study the dynamics of protein aggregation in cellular models of Chorea Huntington and other polyQ diseases, as FLIP gives access to the full spatio-temporal profile of intensity changes in the cell geometry. In contrast to other methods, also dim aggregates become visible during time evolution of fluorescence loss in cellular compartments. However, methods for computational analysis of FLIP data are sparse, and transport models for estimation of transport and diffusion parameters from experimental FLIP sequences are missing.ResultsIn this paper, we present a computational method for analysis of FLIP imaging experiments of intracellular polyglutamine protein aggregates also called inclusion bodies (IBs). By this method, we can determine the diffusion constant and nuclear membrane transport coefficients of polyQ proteins as well as the exchange rates between aggregates and the cytoplasm. Our method is based on a reaction-diffusion multi-compartment model defined on a mesh obtained by segmentation of the cell images from the FLIP sequence. The discontinuous Galerkin (DG) method is used for numerical implementation of our model in FEniCS, which greatly reduces the computing time. The method is applied to representative experimental FLIP sequences, and consistent estimates of all transport parameters are obtained.ConclusionsBy directly estimating the transport parameters from live-cell image sequences using our new computational FLIP approach surprisingly fast exchange dynamics of mutant Huntingtin between cytoplasm and dim IBs could be revealed. This is likely relevant also for other polyQ diseases. Thus, our method allows for quantifying protein dynamics at different stages of the protein aggregation process in cellular models of neurodegeneration.

A Discontinuous Galerkin Model for Fluorescence Loss in Photobleaching

Fluorescence loss in photobleaching (FLIP) is a modern microscopy method for visualization of transport processes in living cells. This paper presents the simulation of FLIP sequences based on a calibrated reaction–diffusion system defined on segmented cell images. By the use of a discontinuous Galerkin method, the computational complexity is drastically reduced compared to continuous Galerkin methods. Using this approach on green fluorescent protein (GFP), we can determine its intracellular diffusion constant, the strength of localized hindrance to diffusion as well as the permeability of the nuclear membrane for GFP passage, directly from the FLIP image series. Thus, we present for the first time, to our knowledge, a quantitative computational FLIP method for inferring several molecular transport parameters in parallel from FLIP image data acquired at commercial microscope systems.

Selective Electrochemical Bleaching of the Outer Leaflet of Fluorescently Labeled Giant Liposomes.

Electrochemistry and confocal fluorescence microscopy were successfully combined to selectively bleach and monitor the fluorescence of NBD (7-nitrobenz-2-oxa-1,3-diazole)-labeled phospholipids of giant liposomes. Three types of giant unilamellar vesicles have been investigated, the fluorescent phospholipids being localized either mainly on their outer-, inner-, or both inner/outer leaflets. We established that only the fluorescent lipids incorporated in the outer leaflet of the vesicles underwent electrochemical bleaching upon reduction. The relative fluorescence intensity decay was quantified all along the electrochemical extinction through an original fluorescence loss in electrobleaching (FLIE) assay. As expected, the reorganization of the fluorescent phospholipids followed diffusion-driven dynamics. This was also evidenced by comparison with fluorescence loss in photobleaching (FLIP) and the corresponding numerical model. The value of the lateral diffusion coefficient of phospholipids was found to be similar to that obtained by other methods reported in the literature. This versatile and selective bleaching procedure appears reliable to explore important biological and pharmacological issues.

Phosphorylation of Human Retinoid X Receptor α at Serine 260 Impairs Its Subcellular Localization, Receptor Interaction, Nuclear Mobility, and 1α,25-Dihydroxyvitamin D3-dependent DNA Binding in Ras-transformed Keratinocytes

Human retinoid X receptor α (hRXRα) plays a critical role in DNA binding and transcriptional activity through heterodimeric association with several members of the nuclear receptor superfamily, including the human vitamin D receptor (hVDR). We previously showed that hRXRα phosphorylation at serine 260 through the Ras-Raf-MAPK ERK1/2 activation is responsible for resistance to the growth inhibitory effects of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), the biologically active metabolite of vitamin D3 To further investigate the mechanism of this resistance, we studied intranuclear dynamics of hVDR and hRXRα-tagged constructs in living cells together with endogenous and tagged protein in fixed cells. We find that hVDR-, hRXRα-, and hVDR-hRXRα complex accumulate in the nucleus in 1α,25(OH)2D3-treated HPK1A cells but to a lesser extent in HPK1ARas-treated cells. Also, by using fluorescence resonance energy transfer (FRET), we demonstrate increased interaction of the hVDR-hRXRα complex in 1α,25(OH)2D3-treated HPK1A but not HPK1ARas cells. In HPK1ARas cells, 1α,25(OH)2D3-induced nuclear localization and interaction of hRXRα are restored when cells are treated with the MEK1/2 inhibitor UO126 or following transfection of the non-phosphorylatable hRXRα Ala-260 mutant. Finally, we demonstrate using fluorescence loss in photobleaching and quantitative co-localization with chromatin that RXR immobilization and co-localization with chromatin are significantly increased in 1α,25(OH)2D3-treated HPK1ARas cells transfected with the non-phosphorylatable hRXRα Ala-260 mutant. This suggests that hRXRα phosphorylation significantly disrupts its nuclear localization, interaction with VDR, intra-nuclear trafficking, and binding to chromatin of the hVDR-hRXR complex.

Temporal regulation of gap junctional communication between tenocytes subjected to static tensile strain with physiological and non-physiological amplitudes

The present study has been performed on temporal changes in gap junctional intercellular communication (GJIC) between tenocytes under static tensile strain with the magnitude of 0% (no strain), 4% (physiological magnitude) or 8% (overloading magnitude) during a 24-h culture period. Tenocytes were isolated from rabbit Achilles tendon and seeded on a stretchable microgroove substrate. GJIC was evaluated as intercellular diffusion coefficient of calcein (DGJ) using fluorescence loss in photobleaching (FLIP) protocol accompanied with a mathematical model of molecular diffusion both within the cell and between the cells. It was exhibited that the application of 4% strain for 1 h increased DGJ significantly. The increased level was maintained for 6 h, followed by returning to the pre-strain level at 24 h. This was associated with a transient increase in connexin 43 (Cx43) gene expression and protein localisation at 1 h, suggesting the increased GJIC may have involved new synthesis of gap junctions. By contrast, the application of 8% static strain reduced DGJ to the similar or lower level from 0% strain group for 6 h, associated with inhibited Cx43 gene expression. However, Cx43 protein localisation was not changed much, and thus, there seem no direct interactions among changes in GJIC, Cx43 gene expression and Cx43 localisation. The present findings highlight the differences in mechanical regulation of GJIC between physiological and non-physiological loadings, and thus the increase or the decrease in GJIC may affect tenocyte functions in different ways.

Compartmentalization of the endoplasmic reticulum in the early C. elegans embryos.

The one-cell Caenorhabditis elegans embryo is polarized to partition fate determinants between the cell lineages generated during its first division. Using fluorescence loss in photobleaching, we find that the endoplasmic reticulum (ER) of the C. elegans embryo is physically continuous throughout the cell, but its membrane is compartmentalized shortly before nuclear envelope breakdown into an anterior and a posterior domain, indicating that a diffusion barrier forms in the ER membrane between these two domains. Using mutants with disorganized ER, we show that ER compartmentalization is independent of the morphological transition that the ER undergoes in mitosis. In contrast, compartmentalization takes place at the position of the future cleavage plane in a par-3-dependent manner. Together, our data indicate that the ER membrane is compartmentalized in cells as diverse as budding yeast, mouse neural stem cells, and the early C. elegans embryo.

Mitochondrial anchorage and fusion contribute to mitochondrial inheritance and quality control in the budding yeast Saccharomyces cerevisiae.

Higher-functioning mitochondria that are more reduced and have less ROS are anchored in the yeast bud tip by the Dsl1-family protein Mmr1p. Here we report a role for mitochondrial fusion in bud-tip anchorage of mitochondria. Fluorescence loss in photobleaching (FLIP) and network analysis experiments revealed that mitochondria in large buds are a continuous reticulum that is physically distinct from mitochondria in mother cells. FLIP studies also showed that mitochondria that enter the bud can fuse with mitochondria that are anchored in the bud tip. In addition, loss of fusion and mitochondrial DNA (mtDNA) by deletion of mitochondrial outer or inner membrane fusion proteins (Fzo1p or Mgm1p) leads to decreased accumulation of mitochondria at the bud tip and inheritance of fitter mitochondria by buds compared with cells with no mtDNA. Conversely, increasing the accumulation and anchorage of mitochondria in the bud tip by overexpression of MMR1 results in inheritance of less-fit mitochondria by buds and decreased replicative lifespan and healthspan. Thus quantity and quality of mitochondrial inheritance are ensured by two opposing processes: bud-tip anchorage by mitochondrial fusion and Mmr1p, which favors bulk inheritance; and quality control mechanisms that promote segregation of fitter mitochondria to the bud.