Cytoskeleton-related proteins in tobacco mycorrhizal cells: gamma-tubulin and clathrin localisation.

Plant cytoskeletal components respond to the penetration of both pathogenic and symbiotic fungi with a new organization of microtubules and microfilaments. To determine the origin and potential role of microtubule arrays previously observed in tobacco cells colonised by an arbuscular mycorrhizal fungus, we have investigated the patterns of gamma-tubulin and clathrin in uninfected and mycorrhizal cells with immunofluorescence techniques. Antibody against gamma-tubulin revealed microtubule organising centers (MTOC) along the nuclear envelope and along the host membrane that surrounds the plant/fungus interface, while clathrin was observed along the peripheral and perifungal membranes, as well as along a tubular system of endomembranes.     

Quantitative imaging of membrane lipid order in cells and organisms

It is now recognized that lipids and proteins in cellular membranes are not homogenously distributed. A high degree of membrane order is the biophysical hallmark of cholesterol-enriched lipid rafts, which may induce the lateral sorting of proteins within the membrane. Here we describe a quantitative fluorescence microscopy technique for imaging localized lipid environments and measuring membrane lipid order in live and fixed cells, as well as in intact tissues. The method is based on the spectral ratiometric imaging of the polarity-sensitive membrane dyes Laurdan and di-4-ANEPPDHQ. Laurdan typically requires multiphoton excitation, making it suitable for the imaging of tissues such as whole, living zebrafish embryos, whereas di-4-ANEPPDHQ imaging can be achieved with standard confocal microscopes. This approach, which takes around 4 h, directly examines the organization of cellular membranes and is distinct from alternative approaches that infer membrane order by measuring probe partitioning or dynamics.     

Analysis of gamete membrane dynamics during double fertilization of Arabidopsis

Angiosperms have a unique sexual reproduction system called “double fertilization.” One sperm cell fertilizes the egg and another sperm cell fertilizes the central cell. To date, plant gamete membrane dynamics during fertilization has been poorly understood. To analyze this unrevealed gamete subcellular behavior, live cell imaging analyses of Arabidopsis double fertilization were performed. We produced female gamete membrane marker lines in which fluorescent proteins conjugated with PIP2a finely visualized egg cell and central cell surfaces. Using those lines together with a sperm cell membrane marker line expressing GCS1-GFP, the double fertilization process was observed. As a result, after gamete fusion, putative sperm plasma membrane GFP signals were occasionally detected on the egg cell surface adjacent to the central cell. In addition, time-lapse imaging revealed that GCS1-GFP signals entered both the egg cell and the central cell in parallel with the sperm cell movement toward the female gametes during double fertilization. These findings suggested that the gamete fusion process based on membrane dynamics was composed of (1) plasma membrane fusion on male and female gamete surfaces, (2) entry of sperm internal membrane components into the female gametes, and (3) plasmogamy.     

GFP as a tool to analyze the organization, dynamics and function of nuclei and microtubules in Neurospora crassa

We report the construction of a versatile GFP expression plasmid and demonstrate its utility in Neurospora crassa. To visualize nuclei and microtubules, we generated carboxy-terminal fusions of sgfp to Neurospora histone H1 (hH1) and beta-tubulin (Bml). Strong expression of GFP fusion proteins was achieved with the inducible Neurospora ccg-1 promoter. Nuclear and microtubule organization and dynamics were observed in live vegetative hyphae, developing asci, and ascospores by conventional and confocal laser scanning fluorescence microscopy. Observations of GFP fusion proteins in live cells largely confirmed previous results obtained by examination of fixed cells with various microscopic techniques. H1-GFP revealed dynamic nuclear shapes. Microtubules were mostly aligned parallel to the growth axis in apical compartments but more randomly arranged in sub-apical compartments. Time-lapse imaging of beta-tubulin-GFP in germinating macroconidia revealed polymerization and depolymerization of microtubules. In heterozygous crosses, H1-GFP and beta-tubulin-GFP expression was silenced, presumably by meiotic silencing. H1-GFP was translated in the vicinity of hH1+-sgfp+ nuclei in the common cytoplasm of giant Banana ascospores, but it diffused into all nuclei, another illustration of the utility of GFP fusion proteins.     

Photoactivation of GFP reveals protein dynamics within the endoplasmic reticulum membrane

Components of the plant cell secretory pathway, including the endoplasmic reticulum and Golgi apparatus, are in constant motion. The photoactivation of GFP has been used to determine that proteins within the membrane of the ER flow as the ER is remodelled. Measurement of the rate at which activated GFP moves away from the activation spot shows that this motion is much faster than would be expected if membrane components moved simply by diffusion. Treatment with latrunculin to depolymerize the actin cytoskeleton stops ER remodelling and reduces the rate of GFP movement to that expected from diffusion alone. This suggests that myosin binds directly or indirectly to ER membrane proteins and actively moves them around over the actin scaffold. Tracking of Golgi body movement was used to demonstrate that they move at the same rate and in the same direction as do photoactivated ER surface proteins. Golgi bodies, therefore, move with, and not over, the surface of the ER. These observations support the current theory of continuity between Golgi bodies and discrete ER exit sites in the ER membrane.     

mEosFP-based green-to-red photoconvertible subcellular probes for plants

Photoconvertible fluorescent proteins (FPs) are recent additions to the biologists' toolbox for understanding the living cell. Like green fluorescent protein (GFP), monomeric EosFP is bright green in color but is efficiently photoconverted into a red fluorescent form using a mild violet-blue excitation. Here, we report mEosFP-based probes that localize to the cytosol, plasma membrane invaginations, endosomes, prevacuolar vesicles, vacuoles, the endoplasmic reticulum, Golgi bodies, mitochondria, peroxisomes, and the two major cytoskeletal elements, filamentous actin and cortical microtubules. The mEosFP fusion proteins are smaller than GFP/red fluorescent protein-based probes and, as demonstrated here, provide several significant advantages for imaging of living plant cells. These include an ability to differentially color label a single cell or a group of cells in a developing organ, selectively highlight a region of a cell or a subpopulation of organelles and vesicles within a cell for tracking them, and understanding spatiotemporal aspects of interactions between similar as well as different organelles. In addition, mEosFP probes introduce a milder alternative to fluorescence recovery after photobleaching, whereby instead of photobleaching, photoconversion followed by recovery of green fluorescence can be used for estimating subcellular dynamics. Most importantly, the two fluorescent forms of mEosFP furnish bright internal controls during imaging experiments and are fully compatible with cyan fluorescent protein, GFP, yellow fluorescent protein, and red fluorescent protein fluorochromes for use in simultaneous, multicolor labeling schemes. Photoconvertible mEosFP-based subcellular probes promise to usher in a much higher degree of precision to live imaging of plant cells than has been possible so far using single-colored FPs.     

Dynamics and mobility of nuclear envelope proteins in interphase and mitotic cells revealed by green fluorescent protein chimeras

Understanding how membrane proteins are targeted to and retained within the nuclear envelope (NE) and the fate of these proteins during NE disassembly/reassembly in mitosis is central for insight into the function of the NE in nuclear organization and dynamics. To address these issues we have attached green fluorescent protein (GFP) to a well-characterized protein of the inner nuclear membrane, lamin B receptor, believed to be one of the major chromatin docking protein in the NE. We have used this construct in a variety of applications, including dual-color GFP time-lapse imaging, to investigate the mechanisms underlying protein targeting to the NE and NE breakdown and reassembly during mitosis. In this review, we present a summary of the results from such studies and discuss the photobleaching and imaging methodology on which they were derived.     

Green light for the secretory pathway

Summary Since the advent of green-fluorescent protein (GFP) technology there has been an explosion of interest in applying this molecule to cell biology. This review summarizes new insights in secretory membrane traffic obtained by the use of GFP fusion proteins. Transport steps between the endoplasmic reticulum and the Golgi apparatus, intra-Golgi traffic, and transport from the Golgi to the plasma membrane are discussed. In addition, insights into the dynamics of the Golgi compartment in plant cells and in mitotic mammalian cells have been included. We conclude that membrane traffic in the secretory pathway appears to be much more dynamic and diverse than previously thought and that GFP promises to be a powerful means to unravel these complex processes.     

Green light for the secretory pathway

Since the advent of green-fluorescent protein (GFP) technology there has been an explosion of interest in applying this molecule to cell biology. This review summarizes new insights in secretory membrane traffic obtained by the use of GFP fusion proteins. Transport steps between the endoplasmic reticulum and the Golgi apparatus, intra-Golgi traffic, and transport from the Golgi to the plasma membrane are discussed. In addition, insights into the dynamics of the Golgi compartment in plant cells and in mitotic mammalian cells have been included. We conclude that membrane traffic in the secretory pathway appears to be much more dynamic and diverse than previously thought and that GFP promises to be a powerful means to unravel these complex processes.     

Zebrafish lines expressing UAS‐driven red probes for monitoring cytoskeletal dynamics

Actin filaments and microtubules are principal components of the cytoskeleton that regulate the basic cellular phenomena underlying many fundamental cellular processes. Therefore, analyzing their dynamics in living cells is important for understanding cellular events more precisely. In this article, we report two novel transgenic zebrafish lines expressing red fluorescent proteins tagged with Lifeact or EB1 that interact with actin filaments and microtubule plus ends, respectively, under the control of the GAL4‐UAS system. Using these transgenic lines, we could detect F‐actin and microtubule plus end dynamics in specific tissues of living zebrafish embryos by crossing with GAL4 driver lines. In addition, we could achieve multi‐color imaging using these transgenic lines with GFP‐expressing transgenic lines. Therefore, our transgenic lines that carry UAS‐driven red fluorescent cytoskeletal probes are useful tools for analyzing spatiotemporal changes of the cytoskeletal elements using multicolor live imaging.     

Making membranes green: construction and characterization of GFP-fusion proteins targeted to discrete plasma membrane domains

Cell membranes contain glycolipid-enriched membrane (GEM) domains, or lipid rafts. GEM domains represent a discrete assembly ofproteins and lipids within the plasma membrane thatfunctions in cell signaling. However, studies of the GEM domains often include the disruption of cells with detergent. Thus, many of the physical and biological properties of GEM domains remain unknown and even controversial. An approach to study these domains but avoid detergent lysis is to measure their properties using the fluorescence imaging of live cells. Accordingly, GFP was targeted to either the GEM or the non-GEMfraction of the plasma membrane using the minimal membrane-anchoring signals of p56lck and pp60c-Src, respectively. The targeting of the fusion proteins to the respective membrane fractions was assayed by membrane fractionation and by quantitating the enrichment in GEM caps in stimulated T cells. The results show that the GEM marker was targeted to GEM domains with similar efficiency as other GEM-associated proteins. Conversely, the non-GEM marker was completely excluded from GEM domains. These constructs represent a useful toolfor studying the discrete fractions of the plasma membrane in live cells using fluorescence imaging.     

Membrane-specific targeting of green fluorescent protein by the Tat pathway in the cyanobacterium Synechocystis PCC6803

The transport and sorting of extracytoplasmic proteins in cyanobacteria is made complex by the presence of a highly differentiated membrane system. Proteins destined for the periplasm and thylakoid lumen are initially transported by Sec- and Tat-type pathways but little is known of the mechanisms that ultimately direct them to the correct destinations. We have generated a Synechocystis PCC6803 transformant that expresses a fusion protein comprising the Tat-specific targeting signal of Escherichia coli TorA linked to green fluorescent protein (GFP). Immunoblotting indicates the presence of mature-size GFP but no precursor form, demonstrating that efficient translocation has taken place. Confocal microscopy and immunogold electron microscopy reveal GFP to be almost exclusively located in the periplasm, with almost no protein evident in the thylakoid network. These data point to the operation of highly effective sorting pathways for soluble proteins in this cyanobacterium. The observed sorting of the GFP suggests that either (a) the Tat apparatus is located only in the plasma membrane or (b) the TorA-GFP is targeted across either membrane but the GFP is subsequently directed to the periplasm, perhaps by a default sorting pathway to this compartment.     

Display of green fluorescent protein on Escherichia coli cell surface

In this study, expression of green fluorescence protein (GFP) on the external surface of Escherichia coli was achieved by construction of a fusion protein between Lpp-OmpA hybrid and a GFP variant, GFPmut2. The GFP was fused in frame to the carboxyl-terminus of Lpp-OmpA fusion previously shown to direct various other heterologous proteins to E. coli cell surface. Western blot analysis of membrane fractions identified the Lpp-OmpA-GFP fusion protein with the expected size (43 kDa). Immunofluorescence microscopy, immunoelectron microscopy, protease and extracellular pH sensitivity assays further confirmed that GFP is anchored on the outer membrane. The GFP displayed on the E. coli outer surface retained its fluorescence and was not susceptible to the indigenous outer membrane protease OmpT even though there are two putative OmpT proteolytic sites present in GFP. Optimization of the expression conditions was conducted using fluorometry, eliminating cumbersome immuno-labeling procedures. Surface-displayed GFP could be used in a variety of applications including screening of polypeptide libraries, development of live vaccines, construction of whole cell allosteric biosensors, and signal transduction studies.     

Aquaporin-4 (AQP4) Associations and Array Dynamics Probed by Photobleaching and Single-molecule Analysis of Green Fluorescent Protein-AQP4 Chimeras

The plasma membrane assembly of aquaporin-4 (AQP4) water channels into orthogonal arrays of particles (OAPs) involves interactions of AQP4 N-terminal domains. To study in live cells the site of OAP assembly, the size and dynamics of plasma membrane OAPs, and the heterotetrameric associations of AQP4, we constructed green fluorescent protein (GFP)-labeled AQP4 “long” (M1) and “short” (M23) isoforms in which GFP was inserted at the cytoplasm-facing N or C terminus or between Val-141 and Val-142 in the second extracellular loop of AQP4. The C-terminal and extracellular loop GFP insertions did not interfere with the rapid unrestricted membrane diffusion of GFP-labeled M1 or the restricted diffusion and OAP assembly of GFP-labeled M23. Photobleaching of brefeldin A-treated cells showed comparable and minimally restricted diffusion of M1 and M23, indicating that OAP assembly occurs post-endoplasmic reticulum. Single-molecule step photobleaching and intensity analysis of GFP-labeled M1 in the absence versus presence of excess unlabeled M1 or M23 with an OAP-disrupting mutation indicated heterotetrameric AQP4 association. Time-lapse total internal reflection fluorescence imaging of M23 in live cells at 37 °C indicated that OAPs diffuse slowly (D ∼ 10⁻¹² cm²/s) and rearrange over tens of minutes. Our biophysical measurements in live cells thus reveal extensive AQP4 monomer-monomer and tetramer-tetramer interactions.     

Simultaneous quantification of cell motility and protein-membrane-association using active contours

We present a new method for the quantification of dynamic changes in fluorescence intensities at the cell membrane of moving cells. It is based on an active contour method for cell-edge detection, which allows tracking of changes in cell shape and position. Fluorescence intensities at specific cortical subregions can be followed in space and time and correlated with cell motility. The translocation of two GFP tagged proteins (CRAC and GRP1) from the cytosol to the membrane in response to stimulation with the chemoattractant cAMP during chemotaxis of Dictyostelium cells and studies of the spatio-temporal dynamics of this process exemplify the method: We show that the translocation can be correlated with motility parameters and that quantitative differences in the rate of association and dissociation from the membrane can be observed for the two PH domain containing proteins. The analysis of periodic CRAC translocation to the leading edge of a cell responding to natural cAMP waves in a mound demonstrates the power of this approach. It is not only capable of tracking the outline of cells within aggregates in front of a noisy background, but furthermore allows the construction of spatio-temporal polar plots, capturing the dynamics of the protein distribution at the cell membrane within the cells' moving co-ordinate system. Compilation of data by means of normalised polar plots is suggested as a future tool, which promises the so-far impossible practicability of extensive statistical studies and automated comparison of complex spatio-temporal protein distribution patterns.     

ARL1 Plays a Role in the Binding of the GRIP Domain of a Peripheral Matrix Protein to the Golgi Apparatus in Plant Cells

ARF GTPases play a central role in regulating membrane dynamics and protein transport in eukaryotic cells. ARF-like (ARL) proteins are close relatives of the ARF regulators of vesicular transport, but their function in plant cells is poorly characterized. Here, by means of live cell imaging and site-directed mutagenesis, we have investigated the cellular function of the plant GTPase ARL1. We provide direct evidence for a role of this ARL family member in the association of a plant golgin with the plant Golgi apparatus. Our data reveal the existence of key residues within the conserved GRIP-domain of the golgin and within the GTPase ARL1 that are central to ARL1–GRIP interaction. Mutations of these residues abolish the interaction of GRIP with the GTP-bound ARL1 and induce a redistribution of GRIP into the cytosol. This indicates that the localization of GRIP to the Golgi apparatus is strongly influenced by the interaction of GRIP with Golgi-localized ARL1. Our results assign a cellular role to a member of the Arabidopsis ARL family in the plant secretory pathway and propose mechanisms for localization of peripheral golgins to the plant Golgi apparatus. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

A novel Golgi-localisation domain shared by a class of coiled-coil peripheral membrane proteins

The mechanism by which peripheral membrane proteins are targeted to the cytoplasmic face of the Golgi apparatus is poorly understood. Previously, we have identified a carboxy-terminal domain of the trans-Golgi-network (TGN) protein p230 that is responsible for Golgi localisation [1]. Here, we report the identification of a similar Golgi-localisation domain (GLD, also termed the 'GRIP' domain - see the paper by Munro and Nichols elsewhere in this issue) in a family of putative peripheral membrane proteins from lower and higher eucaryotes. The majority of family members have a domain structure similar to that of p230, with extensive coiled-coil regions (>80%) and the potential GLD located in a non-coiled-coil domain at the carboxyl terminus. Previously reported proteins in this family include human golgin-97 and Saccharomyces cerevisiae Imh1p. By constructing chimeric cDNAs encoding carboxy-terminal regions of these family members fused to green fluorescent protein (GFP), we have directly demonstrated that the GLD of p230, golgin-97, the newly identified human protein GCC1p and yeast Imh1p functions as a Golgi-targeting domain in transfected mammalian cells. Site-directed mutagenesis of the GLDs identified two conserved aromatic residues that are critical for the function of this targeting domain. Endogenous p230 was displaced from the Golgi membranes in transfected cells expressing high levels of GFP fused to the GLD of either p230 or golgin-97, indicating that different GLDs interact with similar membrane determinants. Thus, we have identified a family of coiled-coil proteins that share a domain shown to be sufficient for the localisation of peripheral membrane proteins to the Golgi apparatus.     

Interaction between cells and elastin fibers: An ultrastructural and immunocytochemical study

Mesenchymal cells (fibroblasts, smooth muscle cells) and endothelial cells were shown to interact with elastin fibers. The strong adhesion of elastin fibers to these cells is mediated by a cell membrane complex with a major glycoprotein component of 120 kDa designated as elastonectin. This interaction was studied by transmission electron microscopy (TEM) and immunocytochemical techniques using antibodies raised against the elastin adhesive proteins. When fibroblasts and smooth muscle cells were cultured in presence of elastin fibers, TEM showed an adhesion mechanism that takes place over several sites along the plasma membrane of these cells. Endothelial cells showed a very close association with elastin, emitting “pseudopodia” that embody the fibers. TEM, indirect immunofluorescence, immunoperoxidase, and confocal microscopy showed the presence and localization of cell membrane components synthesized in large quantities when cells were incubated in presence of elastin. Cells without elastin fibers barely revealed the adhesive membrane complex. These results confirm and extend previous findings concerning the presence of an inducible cell membrane complex that mediates the adhesion of elastin fibers to these cell types. © 1994 Wiley-Liss, Inc.     

Dynamics of membrane clathrin-coated structures during cytokinesis

Remodeling of cell membranes takes place during motile processes such as cell migration and cell division. Defects of proteins involved in membrane dynamics, including clathrin and dynamin, disrupt cytokinesis. To understand the function of clathrin-containing structures (CCS) in cytokinesis, we have expressed a green fluorescent protein (GFP) fusion protein of clathrin light chain a (GFP-clathrin) in NRK epithelial cells and recorded images of dividing cells near the ventral surface with a spinning disk confocal microscope. Punctate GFP-CCS underwent dynamic appearance and disappearance throughout the ventral surface. Following anaphase onset, GFP-CCS between separated chromosomes migrated toward the equator and subsequently disappeared in the equatorial region. Movements outside separating chromosomes were mostly random, similar to what was observed in interphase cells. Directional movements toward the furrow were dependent on both actin filaments and microtubules, while the appearance/disappearance of CCS was dependent on actin filaments but not on microtubules. These results suggest that CCS are involved in remodeling the plasma membrane along the equator during cytokinesis. Clathrin-containing structures may also play a role in transporting signaling or structural components into the cleavage furrow.