Immersed-Prism TIRF Microscopy for Visualizing Intraflagellar Transport in Live Cells

The last stage of the constitutive secretory pathway is the delivery and fusion of post-Golgi transport vesicles (carriers) to the plasma membrane. However, it is still unclear where at the plasma membrane these carriers fuse, in a living cell. In this work we used total internal reflection fluorescence microscopy (TIR-FM) to study the delivery and fusion of post-Golgi carriers, as well as of recycling vesicles from the endosomal compartment, to the cell surface in live cells. TIR-FM is suited to image fluorescent molecules near the cell-substrate interphase, since it selectively illuminates the contact surface of cells. One aim of this work was to establish a quantitative method for the microscopic detection of single exocytic fusion events. Following the localization of fusion we answered basic, previously unresolved, questions in the field of membrane traffic. 1\. Imaging fusion of single post-Golgi carriers. A TIR-FM system was optimized to image exocytosis of single post- Golgi carriers. By labeling the carriers with a GFP-tagged membrane protein (vesicular stomatitis virus glycoprotein, VSVG), quantitative criteria for the microscopic detection of single carrier fusion events were established for the first time. Quantitative analysis of time-lapse images could clearly distinguish fusion of the carriers from both movement of carriers relative to the plasma membrane as well as lysis of carriers. The flattening of the carriers into the plasma membrane as well as the subsequent diffusion of the membrane cargo into the plasma membrane was resolved. The duration of the flattening process was found to depend on the size of the carrier, distinguishing small spherical from large tubular carriers. 2\. Role of microtubules in post-Golgi traffic of fibroblasts. The simul-taneous imaging of post-Golgi carriers and microtubules using a novel dual-color TIR-FM system showed that post-Golgi carriers are transported along microtubules to the fusion sites at the plasma membrane. The data strongly suggested that the carriers are capable of undergoing fusion while still attached to the microtubules and that the carriers do not have to reach the end of the microtubules in order to fuse. In contrast to stationary fibroblasts, migrating fibroblasts were shown to have a microtubule-mediated mechanism for polarized insertion of post-Golgi carriers (here LDL-receptor-GFP) close to the leading edge. Disrupting the microtubules restricted this directed delivery of the carriers to regions of the plasma membrane close to the Golgi complex, making the distribution of fusion sites in stationary and migrating cells indistinguishable. Disrupting the microtubules also decreased the overall fusion frequency, increased the frequency of "partial" fusions, and increased the amount of cargo delivered per fusion. We conclude that the microtubule cytoskeleton is necessary for the domain-specific delivery of post-Golgi membrane cargo in fibroblasts. 3\. Role of microtubules in post- Golgi traffic of polarized epithelial cells. Time-lapse fluorescence microscopy was used to analyze the delivery of apical and basolateral membrane proteins to the cell surface in both non-polarized and polarized epithelial cells. We demonstrated that post-Golgi carriers containing either apical or basolateral membrane proteins fuse to the basal membrane in non-polarized cells. Upon polarization, exocytosis of all carriers to the basal membrane was abrogated. Basolateral carriers were seen to fuse to sites at the lateral membrane, while apical carriers presumably fused to the apical membrane. This selective targeting is concomitant with redistribution of the t-SNAREs, syntaxin 3 and 4, upon polarization. Furthermore, we showed that both the targeted exocytosis of apical proteins and the exclusive localization of syntaxin 3 at the apical plasma membrane are dependent on intact microtubules in polarized epithelial cells. In contrast, targeted exocytosis of basolateral proteins and the basolateral distribution of syntaxin 4 and sec6 are maintained independently of microtubules in polarized cells. 4\. Insulin- regulated recycling of glucose transporter. We studied the insulin-regulated release of the glucose transporter (GLUT4) from the endosomal recycling compartment (ERC) in live cells. We show that GLUT4 is retained within the transferrin receptor-containing general ERC in fibroblasts. Using dual-color TIR-FM, we demonstrate that the transferrin receptor and GLUT4 are transported from the ERC in separate vesicles. This provides the first functional evidence for the formation of distinct classes of vesicles from the ERC. We propose that GLUT4 is dynamically retained within the ERC in fibroblasts because it is concentrated in vesicles that form more slowly than those that transport transferrin receptor.

Force generation by actin assembly shapes cellular membranes. An experimentally constrained multiscale model shows that a minimal branched actin network is sufficient to internalize endocytic pits against membrane tension. Around 200 activated Arp2/3 complexes are required for robust internalization. A newly developed molecule-counting method determined that ~200 Arp2/3 complexes assemble at sites of clathrin-mediated endocytosis in human cells. Simulations predict that actin self-organizes into a radial branched array with growing ends oriented toward the base of the pit. Long actin filaments bend between attachment sites in the coat and the base of the pit. Elastic energy stored in bent filaments, whose presence was confirmed by cryo-electron tomography, contributes to endocytic internalization. Elevated membrane tension directs more growing filaments toward the base of the pit, increasing actin nucleation and bending for increased force production. Thus, spatially constrained actin filament assembly utilizes an adaptive mechanism enabling endocytosis under varying physical constraints.

Dissecting the Molecular Mechanisms of Intraflagellar Transport in Chlamydomonas

Introduction Virulent pathogens hijack host cell machinery during their infection scheme. Commonly, fundamental biochemical processes are discovered through investigation of pathogen infection, especially in the case of pathogen modulation of low abundance lipid species. Ebola virus (EBOV) is a highly virulent, filamentous, lipid-enveloped virus that causes hemorrhagic fever and a fatality rate of 50-90%. The limited understanding of the viral replication in host cells proves difficult for understanding how it spreads. EBOV only has seven genes in its genome, one of which-VP40-is essential for viral egress. VP40 can form virus like particles (VLPs) on its own at the plasma membrane (PM) in live cells and is an excellent model for studying EBOV spread. Objective: We are implementing several assays in live COS-7 cells to decode the PM phospholipid specificity where viral particle formation occurs. Methods: Confocal Laser Scanning Microscopy (CLSM), Spinning Disk and Total Internal Reflection Microscopy (TIRF), VLP Collection. Results: VP40 PM localization can be blocked by elimination of phospholipid substrates through various assays in live COS-7 cells. We have demonstrated that both PS and PI(4,5)P2 are important for VP40 localization and VLP formation at the PM in live cells. We are also investigating the implications on VLP budding as the result of a decrease in PM localization. Conclusions Phospholipid-focused assays in live cells reveal VP40 lipid binding specificity at various cellular sites, other quantitative methods aid in decoding the dynamics and nature of these interactions. Financial Support: NIH AI081077

Total internal reflection fluorescence (TIRF) microscopy (TIRFM) is an elegant optical technique that provides for the excitation of fluorophores in an extremely thin axial region ("optical section"). The method is based on the principle that when excitation light is totally internally reflected in a transparent solid (e.g., coverglass) at its interface with liquid, an electromagnetic field, called the evanescent wave, is generated in the liquid at the solid-liquid interface and is the same frequency as the excitation light. Since the intensity of the evanescent wave exponentially decays with distance from the surface of the solid, only fluorescent molecules within a few hundred nanometers of the solid are efficiently excited. This unit will briefly review the history, optical theory, and different hardware configurations used in TIRFM. In addition, it will provide experimental details and methodological considerations for studying receptors at the plasma membrane in neurons.

The brain contains glial cells. Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons. However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

In Alzheimer disease, amyloid beta, a 39-43-residue peptide produced by cleavage from a large amyloid precursor protein, undergoes conformational change to form amyloid fibrils and deposits as senile amyloid plaques in the extracellular cerebral cortices of the brain. However, the mechanism of how the intrinsically linear amyloid fibrils form spherical senile plaques is unknown. With total internal reflection fluorescence microscopy combined with the use of thioflavin T, an amyloid-specific fluorescence dye, we succeeded in observing the formation of the senile plaque-like spherulitic structures with diameters of around 15 microm on the chemically modified quartz surface. Real-time observation at a single fibrillar level revealed that, in the absence of tight contact with the surface, the cooperative and radial growth of amyloid fibrils from the core leads to a huge spherulitic structure. The results suggest the underlying physicochemical mechanism of senile plaque formation, essential for obtaining insight into prevention of Alzheimer disease.

Total internal reflection fluorescence (TIRF) microscopy has gained popularity in recent years among cell biologists due to its ability to clearly visualize events that occur at the adherent plasma membrane of cells. TIRF microscopy systems are now commercially available from nearly all microscope suppliers. This review aims to give the reader an introduction to the physical basis of TIRF and considerations that need to be made when purchasing a commercial system. We explain how TIRF can be combined with other microscopy modalities and describe how to use TIRF to study processes such as endocytosis, exocytosis, and focal adhesion dynamics. Finally, we provide a step-by-step guide to imaging and analyzing focal adhesion dynamics in a migrating cell using TIRF microscopy.

Whether alpha6beta4 integrin regulates migration remains controversial. beta4 integrin-deficient (JEB) keratinocytes display aberrant migration in that they move in circles, a behavior that mirrors the circular arrays of laminin (LM)-332 in their matrix. In contrast, wild-type keratinocytes and JEB keratinocytes, induced to express beta4 integrin, assemble laminin-332 in linear tracks over which they migrate. Moreover, laminin-332-dependent migration of JEB keratinocytes along linear tracks is restored when cells are plated on wild-type keratinocyte matrix, whereas wild-type keratinocytes show rotation over circular arrays of laminn-332 in JEB keratinocyte matrix. The activities of Rac1 and the actin cytoskeleton-severing protein cofilin are low in JEB keratinocytes compared with wild-type cells but are rescued following expression of wild-type beta4 integrin in JEB cells. Additionally, in wild-type keratinocytes Rac1 is complexed with alpha6beta4 integrin. Moreover, Rac1 or cofilin inactivation induces wild-type keratinocytes to move in circles over rings of laminin-332 in their matrix. Together these data indicate that laminin-332 matrix organization is determined by the alpha6beta4 integrin/actin cytoskeleton via Rac1/cofilin signaling. Furthermore, our results imply that the organizational state of laminin-332 is a key determinant of the motility behavior of keratinocytes, an essential element of skin wound healing and the successful invasion of epidermal-derived tumor cells.

Polarization-Controlled TIRFM with Focal Drift and Spatial Field Intensity Correction

The assembly and maintenance of cilia require intraflagellar transport (IFT), a microtubule-dependent bidirectional motility of multisubunit protein complexes along ciliary axonemes. Defects in IFT and the functions of motile or sensory cilia are associated with numerous human ailments, including polycystic kidney disease and Bardet-Biedl syndrome. Here, we identify a novel Caenorhabditis elegans IFT gene, IFT-associated gene 1 (ifta-1), which encodes a WD repeat-containing protein with strong homology to a mammalian protein of unknown function. Both the C. elegans and human IFTA-1 proteins localize to the base of cilia, and in C. elegans, IFTA-1 can be observed to undergo IFT. IFTA-1 is required for the function and assembly of cilia, because a C. elegans ifta-1 mutant displays chemosensory abnormalities and shortened cilia with prominent ciliary accumulations of core IFT machinery components that are indicative of retrograde transport defects. Analyses of C. elegans IFTA-1 localization/motility along bbs mutant cilia, where anterograde IFT assemblies are destabilized, and in a che-11 IFT gene mutant, demonstrate that IFTA-1 is closely associated with the IFT particle A subcomplex, which is implicated in retrograde IFT. Together, our data indicate that IFTA-1 is a novel IFT protein that is required for retrograde transport along ciliary axonemes.

SummaryFluidity is essential for many biological membrane functions. The basis for understanding membrane structure remains the classic Singer-Nicolson model, in which proteins are embedded within a fluid lipid bilayer and able to diffuse laterally within a sea of lipid. Here we report lipid and protein diffusion in the plasma membrane of live cells of the bacterium Escherichia coli, using Fluorescence Recovery after Photobleaching (FRAP) and Total Internal Reflection Fluorescence (TIRF) microscopy to measure lateral diffusion coefficients. Lipid and protein mobility within the membrane were probed by visualizing an artificial fluorescent lipid and a simple model membrane protein consisting of a single membrane-spanning alpha-helix with a Green Fluorescent Protein (GFP) tag on the cytoplasmic side. The effective viscosity of the lipid bilayer is strongly temperature-dependent, as indicated by changes in the lipid diffusion coefficient. Surprisingly, the mobility of the model protein was unaffected by changes in the effective viscosity of the bulk lipid, and TIRF microscopy indicates that it clusters in segregated, mobile domains. We suggest that this segregation profoundly influences the physical behaviour of the protein in the membrane, with strong implications for bacterial membrane function and bacterial physiology.

Imaging GPCRs trafficking and signaling with total internal reflection fluorescence microscopy in cultured neurons.

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43®) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

TIRF Microscopy Studies of Axial Protein Organization using Photoswitchable Fluorescent Proteins