Nucleation and growth of cadherin adhesions

Cell-cell contact formation relies on the recruitment of cadherin molecules and their anchoring to actin. However, the precise chronology of events from initial cadherin trans-interactions to adhesion strengthening is unclear, in part due to the lack of access to the distribution of cadherins within adhesion zones. Using N-cadherin expressing cells interacting with N-cadherin coated surfaces, we characterized the formation of cadherin adhesions at the ventral cell surface. TIRF and RIC microscopies revealed streak-like accumulations of cadherin along actin fibers. FRAP analysis indicated that engaged cadherins display a slow turnover at equilibrium, compatible with a continuous addition and removal of cadherin molecules within the adhesive contact. Association of cadherin cytoplasmic tail to actin as well as actin cables and myosin II activity are required for the formation and maintenance of cadherin adhesions. Using time lapse microscopy we deciphered how cadherin adhesions form and grow. As lamellipodia protrude, cadherin foci stochastically formed a few microns away from the cell margin. Neo-formed foci coalesced aligned and coalesced with preformed foci either by rearward sliding or gap filling to form cadherin adhesions. Foci experienced collapse at the rear of cadherin adhesions. Based on these results, we present a model for the nucleation, directional growth and shrinkage of cadherin adhesions.     

A transient decrease of electrochemical gradient stabilizes DNA structural change in single mitochondria of living cells

The effect of controlled and reversible perturbation of the electrochemical gradient on the structural changes of mitochondrial DNA has been studied in living cells by fluorescence microscopy. Electrochemical gradient perturbations were induced by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and quantified by measuring the mitochondrial membrane potential using tetramethyl rhodamine methyl ester. Under our experimental conditions, we have shown that ethidium fluorescence was mainly due to ethidium molecules intercalated in mtDNA. Ethidium fluorescence variations have been used to probe DNA structural changes. This showed that: i) electrochemical gradient perturbations induced mtDNA structural change; ii) this change was readily reversible following a total but short collapse of the electrochemical gradient; iii) in contrast, a short and weak perturbation of the electrochemical gradient stabilized the mtDNA structural change; and iv) the degree of weak depolarization varied from cell to cell, showing the necessity of studying the effect of energetic perturbations at the level of an individual cell.     

The formation and stability of DC-SIGN microdomains require its extracellular moiety

Dendritic cell-specific intercellular adhesion molecule (ICAM)-3-grabbing non-integrin (DC-SIGN) is a Ca(2+) -dependent transmembrane lectin that binds a large variety of pathogens and facilitates their uptake for subsequent antigen presentation. This receptor is present in cell surface microdomains, but factors involved in microdomain formation and their exceptional stability are not clear. To determine which domain/motif of DC-SIGN facilitates its presence in microdomains, we studied mutations at key locations including truncation of the cytoplasmic tail, and ectodomain mutations that resulted in the removal of the N-linked glycosylation site, the tandem repeats and the carbohydrate recognition domain (CRD), as well as modification of the calcium sites in the CRD required for carbohydrate binding. Confocal imaging and fluorescence recovery after photobleaching measurements showed that the cytoplasmic domain and the N-linked glycosylation site do not affect the ability of DC-SIGN to form stable microdomains. However, truncation of the CRD results in complete loss of visible microdomains and subsequent lateral diffusion of the mutants. Apart from cell adhesions, membrane domains are thought to be localized primarily via the cytoskeleton. By contrast, we propose that interactions between the CRD of DC-SIGN and the extracellular matrix and/or cis interactions with transmembrane scaffolding protein(s) play an essential role in organizing these microdomains.     

A novel fluorescent probe with large Stokes shift for the detection of viscosity changes and its imaging in living cells

As an important cellular microenvironmental parameter, viscosity could reflect the status of living cells. Small molecular fluorescent probes are a vital tool to measure the change of viscosity in living cells. A novel fluorescence probe ZL-1 with a large Stokes shift (in methanol it reached to 153 nm and in glycerol it reached to 125 nm) and excellent sensitivity toward viscosity was developed. The sharp enhancement of the emission intensity for the probe ZL-1 from low viscous methanol to high viscous glycerol indicated that the probe ZL-1 could respond to the viscosity variations. Moreover, the probe ZL-1 has been successfully utilized to detect of the viscosity variations in living cells.     

Retention and mobility of the mammalian lamin B receptor in the plant nuclear envelope

Background information. In a previous study, we showed that GFP (green fluorescent protein) fused to the N‐terminal 238 amino acids of the mammalian LBR (lamin B receptor) localized to the NE (nuclear envelope) when expressed in the plant Nicotiana tabacum. The protein was located in the NE during interphase and migrated with nuclear membranes during cell division. Targeting and retention of inner NE proteins requires several mechanisms: signals that direct movement through the nuclear pore complex, presence of a transmembrane domain or domains and retention by interaction with nuclear or nuclear‐membrane constituents. Results. Binding mutants of LBR—GFP were produced to investigate the mechanisms for the retention of LBR in the NE. FRAP (fluorescence recovery after photobleaching) analysis of mutant and wild‐type constructs was employed to examine the retention of LBR—GFP in the plant NE. wtLBR—GFP (wild‐type LBR—GFP) was shown to have significantly lower mobility in the NE than the lamin‐binding domain deletion mutant, which showed increased mobility in the NE and was also localized to the endoplasmic reticulum and punctate structures in some cells. Modification of the chromatin‐binding domain resulted in the localization of the protein in nuclear inclusions, in which it was immobile. Conclusions. As expression of truncated LBR—GFP in plant cells results in altered targeting and retention compared with wtLBR—GFP, we conclude that plant cells can recognize the INE (inner NE)‐targeting motif of LBR. The altered mobility of the truncated protein suggests that not only do plant cells recognize this signal, but also have nuclear proteins that interact weakly with LBR.     

The dynamics of pre-mRNAs and poly(A)+ RNA at speckles in living cells revealed by iFRAP studies

Speckles are subnuclear domains where pre-mRNA splicing factors accumulate in the interchromatin space. To investigate the dynamics of mRNAs at speckles, fluorescently labeled Drosophila Fushitarazu (ftz) pre-mRNAs were microinjected into the nuclei of Cos7 cells and the dissociation kinetics of pre-mRNAs from speckles was analyzed using photobleaching techniques. The microinjected ftz pre-mRNAs accumulated in speckles in an intron-dependent manner and were spliced and exported to the cytoplasm with a half-time of about 10 min. Dissociation of the accumulated pre-mRNAs in speckles exhibited rapid diffusion and slow-dissociation of about 100 s. The slow-dissociation required metabolic energy of ATP. Two types of splice-defective mutated mRNAs dissociated from the speckle with a time constant similar to that of wild-type mRNA, indicating that slow-dissociation was not coupled to the splicing reaction. Furthermore, some pre-mRNAs shuttled between speckles and nucleoplasm, suggesting that pre-mRNAs repeatedly associated with and dissociated from speckles until introns were removed. Next, endogenous poly(A)+ RNA was visualized by injecting Cy3-labeled 2'O-methyl oligo(U)22 probes. Some poly(A)+ RNA distributed diffusely within the nucleus, but some of them accumulated in speckles and dissociated at time constant of about 100 s.     

Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C- and N-termini of the fimbrin actin-binding domain 2

The role of the actin cytoskeleton in plant development is intimately linked to its dynamic behavior. Therefore it is essential to continue refining methods for studying actin organization in living plant cells. The discovery of green fluorescent protein (GFP) has popularized the use of translational fusions of GFP with actin filament (F-actin) side-binding proteins to visualize in vivo actin organization in plants. The most recent of these live cell F-actin reporters are GFP fusions to the actin-binding domain 2 (ABD2) of Arabidopsis fimbrin 1 (ABD2-GFP). To improve ABD2-GFP fluorescence for enhanced in vivo F-actin imaging, transgenic Arabidopsis plants were generated expressing a construct with GFP fused to both the C- and N-termini of ABD2 under the control of the CaMV 35S promoter (35S::GFP-ABD2-GFP). The 35S::GFP-ABD2-GFP lines had significantly increased fluorescence compared with the original 35S::ABD2-GFP lines. The enhanced fluorescence of the 35S::GFP-ABD2-GFP-expressing lines allowed the acquisition of highly resolved images of F-actin in different plant organs and stages of development because of the reduced confocal microscope excitation settings needed for data collection. This simple modification to the ABD2-GFP construct presents an important tool for studying actin function during plant development.     

Effects of Carbon and Nitrogen Sources on the Levels of Several NADP- and NAD-Linked Dehydrogenase Activities of Hydrocarbon-utilizable Candida Yeasts

The variation of activities of several NADP-linked and NAD-linked dehydrogenases were studied during the aerobic growth of two species of hydrocarbon-utilizable Candida yeasts on different carbon and nitrogen sources. The level of NADP-linked isocitrate dehydrogenase in C. tropicalis and C. lipolytica growing on acetate was significantly higher than that in the yeasts growing on glucose. The glucose-grown cells of C. tropicalis showed a high activity of glucose-6-phosphate dehydrogenase as compared with the acetate-grown cells, while the enzyme level in C. lipolytica was low regardless of carbon sources used. The cells of both yeasts growing on n-alkane and oleic acid contained relatively low activity of NADP-linked isocitrate dehydrogenase. Presence of ion in the acetate medium increased the level of NADP-linked isocitrate dehydrogenase activity. These results suggest that different types of NADPH-generating systems operate alternatively in these yeasts depending upon carbon and nitrogen sources.     

Cholesterol depletion induces dynamic confinement of the G-protein coupled serotonin1A receptor in the plasma membrane of living cells

Cholesterol is an essential constituent of eukaryotic membranes and plays a crucial role in membrane organization, dynamics, function, and sorting. It is often found distributed non-randomly in domains or pools in biological and model membranes and is thought to contribute to a segregated distribution of membrane constituents. Signal transduction events mediated by seven transmembrane domain G-protein coupled receptors (GPCRs) are the primary means by which cells communicate with and respond to their external environment. We analyzed the role of cholesterol in the plasma membrane organization of the G-protein coupled serotonin_1A receptor by fluorescence recovery after photobleaching (FRAP) measurements with varying bleach spot sizes. Our results show that lateral diffusion parameters of serotonin_1A receptors in normal cells are consistent with models describing diffusion of molecules in a homogenous membrane. Interestingly, these characteristics are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin_1A receptors in the plasma membrane. Importantly, analysis of ligand binding and downstream signaling of the serotonin_1A receptor suggests that receptor function is affected in a significantly different manner when intact cells or isolated membranes are depleted of cholesterol. These results assume significance in the context of interpreting effects of cholesterol depletion on diffusion characteristics of membrane proteins in particular, and cholesterol-dependent cellular processes in general.     

Specific lectins map the distribution of fibronectin and beta 1-integrin on living MDCK cells

The expression and dynamics of bound fibronectin and the sialylated integral membrane protein, beta 1-integrin, were analyzed on the apical membrane of living MDCK cells. Fibronectin was identified by its specific binding of fluorescent peanut agglutinin and sialylated beta 1-integrin by its binding of Sambucus nigra agglutinin. Confocal epifluorescence microscopy and laser scanning cytometry determined the distribution and abundance of binding sites of the two fluorescently labeled lectins. Both fibronectin and beta 1-integrin were restricted to specific regions uniformly distributed over the entire apical surface. Apical-surface fibronectin binding varied much more between cells than did the expression of beta 1-integrin. Sialylated beta 1-integrin colocalized >92% with membrane microplicae while fibronectin was unrelated to these surface structures. This lack of colocalization of the proteins was confirmed by double-labeling experiments. From the maturation dependence of the fibronectin-binding capacity and the differences in protein turnover times, it was evident that fibronectin did not bind to sialylated beta 1-integrin. Furthermore, desialylation of beta 1-integrin uncovered additional fibronectin receptors on the apical membrane. We conclude that these lectins permit tracking of two membrane-associated glycoproteins in living cells and that fibronectin binds only to desialylated beta 1-integrin on MDCK cells.     

Determination of protein mobility in mitochondrial membranes of living cells

Molecular mobility in membranes of intracellular organelles is poorly understood, due to the lack of experimental tools applicable for a great diversity of shapes and sizes such organelles can acquire. Determinations of diffusion within the plasma membrane or cytosol are based mostly on the assumption of an infinite flat space, not valid for curved membranes of smaller organelles. Here we extend the application of FRAP to mitochondria of living cells by application of numerical analysis to data collected from a small region inside a single organelle. The spatiotemporal pattern of light pulses generated by the laser scanning microscope during the measurement is reconstructed in silico and consequently the values of diffusion parameters best suited to the particular organelle are found. The mobility of the outer membrane proteins hFis and Tom7, as well as oxidative phosphorylation complexes COX and F₁F₀ ATPase located in the inner membrane is analyzed in detail. Several alternative models of diffusivity applied to these proteins provide insight into the mechanisms determining the rate of motion in each of the membranes. Tom7 and hFis move along the mitochondrial axis in the outer membrane with similar diffusion coefficients (D = 0.7 μm²/s and 0.6 μm²/s respectively) and equal immobile fraction (7%). The notably slower motion of the inner membrane proteins is best represented by a dual-component model with approximately equal partitioning of the fractions (F₁F₀ ATPase: 0.4 μm²/s and 0.0005 μm²/s; COX: 0.3 μm²/s and 0.007 μm²/s). The mobility patterns specific for the membranes of this organelle are unambiguously distinguishable from those of the plasma membrane or artificial lipid environments: The parameters of mitochondrial proteins indicate a distinct set of factors responsible for their diffusion characteristics.     

Vault mobility depends in part on microtubules and vaults can be recruited to the nuclear envelope

Vaults are ribonucleoproteins that may function in intracellular transport processes. We investigated the intracellular distribution and dynamics of vaults in non-small cell lung cancer cells in which vaults are labeled with the green fluorescent protein. Immunofluorescence experiments showed that vaults are dispersed throughout the cytoplasm; a small fraction is found in close proximity to microtubules. Immunoprecipitation experiments corroborated these results showing co-precipitation of MVP and beta-tubulin. Using quantitative fluorescence-recovery after photobleaching (FRAP), we demonstrated that vault mobility over longer distances in part depends on intact microtubules; vaults moving slower when microtubules are depolymerized by nocodazole. Biochemical fractionation indicated a small fraction of MVP associated with the nucleus, however, no GFP-tagged vaults could be observed inside the nucleus. We observed an accumulation of vaults at the nuclear envelope upon treatment of cells with the protein synthesis inhibitor cycloheximide. Analysis of nucleo-cytoplasmic transport using a fluorescent substrate containing a classical NLS and NES expressed in MVP+/+ and MVP-/- mouse embryonic fibroblasts indicated no differences in nuclear import/export kinetics, suggesting no role for vaults in these processes. We hypothesize that a subset of vaults moves directionally via microtubules, possibly towards the nucleus.     

Characterization of BARD1 targeting and dynamics at the centrosome: the role of CRM1, BRCA1 and the Q564H mutation

BARD1 heterodimerizes with BRCA1, forming an E3 ubiquitin ligase that functions at nuclear foci to repair DNA damage and the centrosome to regulate mitosis. We compared BARD1 recruitment at these structures using fluorescence recovery after photobleaching assays to measure YFP-BARD1 dynamics in live cells. In nuclei at ionizing radiation-induced foci, 20% of the BARD1 pool was immobile and 80% of slow mobility exhibiting a recovery time > 500 s. In contrast, at centrosomes 83% of BARD1 was rapidly mobile with extremely fast turnover (recovery time ~ 20 s). The ~ 25-fold faster exchange of BARD1 at centrosomes correlated with BRCA1-independent recruitment. We mapped key targeting sequences to a combination of the N and C-termini, and showed that mutation of the nuclear export signal reduced centrosome localization by 50%, revealing a role for CRM1. Deletion of the sequence 128-550 increased BARD1 turnover at the centrosome, consistent with a role in transient associations. Conversely, the cancer mutation Q564H reduced turnover by 25%. BARD1 is one of the most highly mobile proteins yet detected at the centrosome, and in contrast to its localization at DNA repair foci, which requires dimerization with BRCA1, targeting of BARD1 to the centrosome occurs prior to heterodimerization and its rapid turnover may provide a mechanism to regulate dimer formation.     

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life from embryonic development and wound healing to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell’s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.     

活体生物发光与荧光成像技术在干细胞研究中的应用

活体生物发光与荧光成像技术是近年发展起来的新兴技术,以其操作简便、灵敏度高、创伤性小在生命科学研究中有着较大的优势,目前已被广泛应用于基因标记、细胞凋亡、免疫细胞研究、肿瘤转移等诸多领域,尤其在新兴的干细胞研究方面更是发挥着不可替代的作用。综述了活体生物发光与荧光成像技术的原理、优势、应用范围及发展前景,特别对近年来该技术在胚胎干细胞的肝向分化、人造血干细胞重建小鼠造血系统、神经祖细胞治疗中枢神经系统肿瘤等方面的应用做了详细介绍。     

Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells

In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of <100?nm in diameter in the plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane.     

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either ¹²⁵I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10⁷ binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37°C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.