Golgi Tubule Traffic and the Effects of Brefeldin A Visualized in Living Cells

The Golgi complex is a dynamic organelle engaged in both secretory and retrograde membrane traffic. Here, we use green fluorescent protein–Golgi protein chimeras to study Golgi morphology in vivo. In untreated cells, membrane tubules were a ubiquitous, prominent feature of the Golgi complex, serving both to interconnect adjacent Golgi elements and to carry membrane outward along microtubules after detaching from stable Golgi structures. Brefeldin A treatment, which reversibly disassembles the Golgi complex, accentuated tubule formation without tubule detachment. A tubule network extending throughout the cytoplasm was quickly generated and persisted for 5–10 min until rapidly emptying Golgi contents into the ER within 15–30 s. Both lipid and protein emptied from the Golgi at similar rapid rates, leaving no Golgi structure behind, indicating that Golgi membranes do not simply mix but are absorbed into the ER in BFA-treated cells. The directionality of redistribution implied Golgi membranes are at a higher free energy state than ER membranes. Analysis of its kinetics suggested a mechanism that is analogous to wetting or adsorptive phenomena in which a tension-driven membrane flow supplements diffusive transfer of Golgi membrane into the ER. Such nonselective, flow-assisted transport of Golgi membranes into ER suggests that mechanisms that regulate retrograde tubule formation and detachment from the Golgi complex are integral to the existence and maintenance of this organelle.     

Characterization of a Novel Yeast SNARE Protein Implicated in Golgi Retrograde Traffic

The protein trafficking machinery of eukaryotic cells is employed for protein secretion and for the localization of resident proteins of the exocytic and endocytic pathways. Protein transit between organelles is mediated by transport vesicles that bear integral membrane proteins (v-SNAREs) which selectively interact with similar proteins on the target membrane (t-SNAREs), resulting in a docked vesicle. A novel Saccharomyces cerevisiae SNARE protein, which has been termed Vti1p, was identified by its sequence similarity to known SNAREs. Vti1p is a predominantly Golgi-localized 25-kDa type II integral membrane protein that is essential for yeast viability. Vti1p can bind Sec17p (yeast SNAP) and enter into a Sec18p (NSF)-sensitive complex with the cis-Golgi t-SNARE Sed5p. This Sed5p/Vti1p complex is distinct from the previously described Sed5p/Sec22p anterograde vesicle docking complex. Depletion of Vti1p in vivo causes a defect in the transport of the vacuolar protein carboxypeptidase Y through the Golgi. Temperature-sensitive mutants of Vti1p show a similar carboxypeptidase Y trafficking defect, but the secretion of invertase and gp400/hsp150 is not significantly affected. The temperature-sensitive vti1 growth defect can be rescued by the overexpression of the v-SNARE, Ykt6p, which physically interacts with Vti1p. We propose that Vti1p, along with Ykt6p and perhaps Sft1p, acts as a retrograde v-SNARE capable of interacting with the cis-Golgi t-SNARE Sed5p.     

The Movement of Coiled Bodies Visualized in Living Plant Cells by the Green Fluorescent Protein

Coiled bodies are nuclear organelles that contain components of at least three RNA-processing pathways: pre-mRNA splicing, histone mRNA 3'- maturation, and pre-rRNA processing. Their function remains unknown. However, it has been speculated that coiled bodies may be sites of splicing factor assembly and/or recycling, play a role in histone mRNA 3'-processing, or act as nuclear transport or sorting structures. To study the dynamics of coiled bodies in living cells, we have stably expressed a U2B"-green fluorescent protein fusion in tobacco BY-2 cells and in Arabidopsis plants. Time-lapse confocal microscopy has shown that coiled bodies are mobile organelles in plant cells. We have observed movements of coiled bodies in the nucleolus, in the nucleoplasm, and from the periphery of the nucleus into the nucleolus, which suggests a transport function for coiled bodies. Furthermore, we have observed coalescence of coiled bodies, which suggests a mechanism for the decrease in coiled body number during the cell cycle. Deletion analysis of the U2B" gene construct has shown that the first RNP-80 motif is sufficient for localization to the coiled body.     

Partitioning of the Golgi Apparatus during Mitosis in Living HeLa Cells

The Golgi apparatus of HeLa cells was fluorescently tagged with a green fluorescent protein (GFP), localized by attachment to the NH₂-terminal retention signal of N-acetylglucosaminyltransferase I (NAGT I). The location was confirmed by immunogold and immunofluorescence microscopy using a variety of Golgi markers. The behavior of the fluorescent Golgi marker was observed in fixed and living mitotic cells using confocal microscopy. By metaphase, cells contained a constant number of Golgi fragments dispersed throughout the cytoplasm. Conventional and cryoimmunoelectron microscopy showed that the NAGT I–GFP chimera (NAGFP)-positive fragments were tubulo-vesicular mitotic Golgi clusters. Mitotic conversion of Golgi stacks into mitotic clusters had surprisingly little effect on the polarity of Golgi membrane markers at the level of fluorescence microscopy. In living cells, there was little self-directed movement of the clusters in the period from metaphase to early telophase. In late telophase, the Golgi ribbon began to be reformed by a dynamic process of congregation and tubulation of the newly inherited Golgi fragments. The accuracy of partitioning the NAGFP-tagged Golgi was found to exceed that expected for a stochastic partitioning process. The results provide direct evidence for mitotic clusters as the unit of partitioning and suggest that precise regulation of the number, position, and compartmentation of mitotic membranes is a critical feature for the ordered inheritance of the Golgi apparatus.     

Resveratrol Modulates Mitochondria Dynamics in Replicative Senescent Yeast Cells

Mitochondria form a reticulum network dynamically fuse and divide in the cell. The balance between mitochondria fusion and fission is correlated to the shape, activity and integrity of these pivotal organelles. Resveratrol is a polyphenol antioxidant that can extend life span in yeast and worm. This study examined mitochondria dynamics in replicative senescent yeast cells as well as the effects of resveratrol on mitochondria fusion and fission. Collecting cells by biotin-streptavidin sorting method revealed that majority of the replicative senescent cells bear fragmented mitochondrial network, indicating mitochondria dynamics favors fission. Resveratrol treatment resulted in a reduction in the ratio of senescent yeast cells with fragmented mitochondria. The readjustment of mitochondria dynamics induced by resveratrol likely derives from altered expression profiles of fusion and fission genes. Our results demonstrate that resveratrol serves not only as an antioxidant, but also a compound that can mitigate mitochondria fragmentation in replicative senescent yeast cells.     

Dynamics of Nuclear Pore Distribution in Nucleoporin Mutant Yeast Cells

To follow the dynamics of nuclear pore distribution in living yeast cells, we have generated fusion proteins between the green fluorescent protein (GFP) and the yeast nucleoporins Nup49p and Nup133p. In nup133⁻ dividing cells that display a constitutive nuclear pore clustering, in vivo analysis of GFP-Nup49p localization revealed changes in the distribution of nuclear pore complex (NPC) clusters. Furthermore, upon induction of Nup133p expression in a GAL-nup133 strain, a progressive fragmentation of the NPC aggregates was observed that in turn led to a wild-type nuclear pore distribution. To try to uncouple Nup133p- induced NPC redistribution from successive nuclear divisions and nuclear pore biogenesis, we devised an assay based on the formation of heterokaryons between nup133⁻ mutants and cells either expressing or overexpressing Nup133p. Under these conditions, the use of GFP-Nup133p and GFP-Nup49p fusion proteins revealed that Nup133p can be rapidly targeted to the clustered nuclear pores, where its amino-terminal domain is required to promote the redistribution of preexisting NPCs.     

Visualization of MicrotubuleOrganization and Dynamics in Living Arabidopsis Embryonic Cells

Dear Editor, Embryogenesis is a critical developmental stage during the life cycle of flowering plants. During embryogenesis, the first round of asymmetric cell division in the zygote is followed by a series of cellular events, including cell division （symmetric or asymmetric） and directional cell expan- sion to generate the apical-basal axis, radial and lateral symmetry, and patterns of different cell fates for initiation of different organ primordia, which lay the foundation for post-embryonic development （Jurgens, 2001; Wendrich and Weijers, 2013）.     

Calcium Dynamics in Root Cells of Arabidopsis thaliana Visualized with Selective Plane Illumination Microscopy

Selective Plane Illumination Microscopy (SPIM) is an imaging technique particularly suited for long term in-vivo analysis of transparent specimens, able to visualize small organs or entire organisms, at cellular and eventually even subcellular resolution. Here we report the application of SPIM in Calcium imaging based on Förster Resonance Energy Transfer (FRET). Transgenic Arabidopsis plants expressing the genetically encoded-FRET-based Ca²⁺ probe Cameleon, in the cytosol or nucleus, were used to demonstrate that SPIM enables ratiometric fluorescence imaging at high spatial and temporal resolution, both at tissue and single cell level. The SPIM-FRET technique enabled us to follow nuclear and cytosolic Ca²⁺ dynamics in Arabidopsis root tip cells, deep inside the organ, in response to different stimuli. A relevant physiological phenomenon, namely Ca²⁺ signal percolation, predicted in previous studies, has been directly visualized.     

Photobleaching Assays (FRAP & FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein''s mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression¹. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells^2,3.Download video file.^{(59M, mov)}     

Distribution and Dynamics of Gap Junction Channels Revealed in Living Cells

To study the structural composition and dynamics of gap junctions in living cells, we tagged their subunit proteins, termed connexins, with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. Tagged connexins assembled normally and channels were functional. High-resolution fluorescence images of gap junction plaques assembled from CFP and YFP tagged connexins revealed that the mode of channel distribution is strictly dependent on the connexin isoforms. Co-distribution as well as segregation into well-separated domains was observed. Based on accompanying studies we propose that channel distribution is regulated by intrinsic, connexin isoform specific signals. High-resolution time-lapse images revealed that gap junctions, contrary to previous expectations, are dynamic assemblies of channels. Channels within clusters and clusters themselves are mobile and constantly undergo structural rearrangements. Movements are complex and allow channels to move, comparable to other plasma membrane proteins not anchored to cytoskeletal elements. Comprehensive analysis, however, demonstrated that gap junction channel movements are not driven by diffusion described to propel plasma membrane protein movement. Instead, recent studies suggest that movements of gap junction channels are indirect and predominantly propelled by plasma membrane lipid flow that results from metabolic endo- and exocytosis.     

Distribution and Dynamics of Gap Junction Channels Revealed in Living Cells

To study the structural composition and dynamics of gap junctions in living cells, we tagged their subunit proteins, termed connexins, with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. Tagged connexins assembled normally and channels were functional. High-resolution fluorescence images of gap junction plaques assembled from CFP and YFP tagged connexins revealed that the mode of channel distribution is strictly dependent on the connexin isoforms. Co-distribution as well as segregation into well-separated domains was observed. Based on accompanying studies we propose that channel distribution is regulated by intrinsic, connexin isoform specific signals. High-resolution time-lapse images revealed that gap junctions, contrary to previous expectations, are dynamic assemblies of channels. Channels within clusters and clusters themselves are mobile and constantly undergo structural rearrangements. Movements are complex and allow channels to move, comparable to other plasma membrane proteins not anchored to cytoskeletal elements. Comprehensive analysis, however, demonstrated that gap junction channel movements are not driven by diffusion described to propel plasma membrane protein movement. Instead, recent studies suggest that movements of gap junction channels are indirect and predominantly propelled by plasma membrane lipid flow that results from metabolic endo- and exocytosis.     

COPI-independent Anterograde Transport: Cargo-selective ER to Golgi Protein Transport in Yeast COPI Mutants

The coatomer (COPI) complex mediates Golgi to ER recycling of membrane proteins containing a dilysine retrieval motif. However, COPI was initially characterized as an anterograde-acting coat complex. To investigate the direct and primary role(s) of COPI in ER/Golgi transport and in the secretory pathway in general, we used PCR-based mutagenesis to generate new temperature-conditional mutant alleles of one COPI gene in Saccharomyces cerevisiae, SEC21 (γ-COP). Unexpectedly, all of the new sec21 ts mutants exhibited striking, cargo-selective ER to Golgi transport defects. In these mutants, several proteins (i.e., CPY and α-factor) were completely blocked in the ER at nonpermissive temperature; however, other proteins (i.e., invertase and HSP150) in these and other COPI mutants were secreted normally. Nearly identical cargo-specific ER to Golgi transport defects were also induced by Brefeldin A. In contrast, all proteins tested required COPII (ER to Golgi coat complex), Sec18p (NSF), and Sec22p (v-SNARE) for ER to Golgi transport. Together, these data suggest that COPI plays a critical but indirect role in anterograde transport, perhaps by directing retrieval of transport factors required for packaging of certain cargo into ER to Golgi COPII vesicles. Interestingly, CPY–invertase hybrid proteins, like invertase but unlike CPY, escaped the sec21 ts mutant ER block, suggesting that packaging into COPII vesicles may be mediated by cis-acting sorting determinants in the cargo proteins themselves. These hybrid proteins were efficiently targeted to the vacuole, indicating that COPI is also not directly required for regulated Golgi to vacuole transport. Additionally, the sec21 mutants exhibited early Golgi-specific glycosylation defects and structural aberrations in early but not late Golgi compartments at nonpermissive temperature. Together, these studies demonstrate that although COPI plays an important and most likely direct role both in Golgi–ER retrieval and in maintenance/function of the cis-Golgi, COPI does not appear to be directly required for anterograde transport through the secretory pathway.     

Nuclear Membrane Dynamics and Reassembly in Living Cells: Targeting of an Inner Nuclear Membrane Protein in Interphase and Mitosis

The mechanisms of localization and retention of membrane proteins in the inner nuclear membrane and the fate of this membrane system during mitosis were studied in living cells using the inner nuclear membrane protein, lamin B receptor, fused to green fluorescent protein (LBR–GFP). Photobleaching techniques revealed the majority of LBR–GFP to be completely immobilized in the nuclear envelope (NE) of interphase cells, suggesting a tight binding to heterochromatin and/or lamins. A subpopulation of LBR–GFP within ER membranes, by contrast, was entirely mobile and diffused rapidly and freely (D = 0.41 ± 0.1 μm²/s). High resolution confocal time-lapse imaging in mitotic cells revealed LBR–GFP redistributing into the interconnected ER membrane system in prometaphase, exhibiting the same high mobility and diffusion constant as observed in interphase ER membranes. LBR–GFP rapidly diffused across the cell within the membrane network defined by the ER, suggesting the integrity of the ER was maintained in mitosis, with little or no fragmentation and vesiculation. At the end of mitosis, nuclear membrane reformation coincided with immobilization of LBR–GFP in ER elements at contact sites with chromatin. LBR–GFP–containing ER membranes then wrapped around chromatin over the course of 2–3 min, quickly and efficiently compartmentalizing nuclear material. Expansion of the NE followed over the course of 30–80 min. Thus, selective changes in lateral mobility of LBR–GFP within the ER/NE membrane system form the basis for its localization to the inner nuclear membrane during interphase. Such changes, rather than vesiculation mechanisms, also underlie the redistribution of this molecule during NE disassembly and reformation in mitosis.     

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a Förster resonance energy transfer (FRET) probe in which the ε subunit of the F_oF₁-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the ε subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.     

Organelle-Specific Nitric Oxide Detection in Living Cells via HaloTag Protein Labeling

Nitric oxide (NO) is a membrane-permeable signaling molecule that is constantly produced, transferred, and consumed in vivo. NO participates and plays important roles in multiple biological processes. However, spatiotemporal imaging of NO in living cells is challenging. To fill the gap in currently used techniques, we exploited the versatility of HaloTag technology and synthesized a novel organelle-targetable fluorescent probe called HTDAF-2DA. We demonstrate the utility of the probe by monitoring subcellular NO dynamics. The developed strategy enables precise determination of local NO function.     

Dielectric Properties of Yeast Cells Expressed With the Motor Protein Prestin

We report on the linear and nonlinear dielectric properties of budding yeast (S. cerevisiae) cells, one strain of which has been genetically modified to express prestin. This motor protein plays a crucial role in the large electromotility exhibited by the outer hair cells of mammalian inner ears. Live cell suspensions exhibit enormous dielectric responses, which can be used to probe metabolic activity, membrane potential, and other properties. The aims of this study are: (1) to compare the dielectric responses of organisms expressing prestin from those of control specimens, and (2) ultimately to further develop dielectric response as a tool to study live cells, proteins, and lipids.     

活细胞钙动态的共聚焦扫描显微镜检测技术

共聚焦激光扫描显微镜（Confocal Laser Scarming Microscope,CLSM）广泛应用于活细胞内钙敏感探针标记的钙水平的动态测量。较之传统的显微镜CLSM在钙成像分析上有着不可比拟的优越性,但也存在一些缺陷,近些年陆续出现了一些针对这些缺陷的改善措施,如比率法、葡聚糖探针及其他一些新技术与共聚焦显微镜的联合应用等,并且出现了诸如双光子显微镜等新型激光共聚焦显微镜。随着共聚焦钙成像技术的不断发展进步,其今后的应用前景将会越越广阔。