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The apical glycoprotein gp135 is delivered to a ring at the base of the primary cilium and subsequently moves in a radial fashion away from the cilium in a microtubule-dependent manner., In polarized epithelial cells, newly synthesized cell surface proteins travel in carrier vesicles from the trans Golgi network to the apical or basolateral plasma membrane. Despite extensive research on polarized trafficking, the sites of protein delivery are not fully characterized. Here we use the SNAP tag system to examine the site of delivery of the apical glycoprotein gp135. We show that a cohort of gp135 is delivered to a ring surrounding the base of the primary cilium, followed by microtubule-dependent radial movement away from the cilium. Delivery to the periciliary ring was specific to newly synthesized and not recycling protein. A subset of this newly delivered protein traverses the basolateral membrane en route to the apical membrane. Crumbs3a, another apical protein, was not delivered to the periciliary region, instead making its initial apical appearance in a pattern that resembled its steady-state distribution. Our results demonstrate a surprising “hot spot” for gp135 protein delivery at the base of the primary cilium and suggest the existence of a novel microtubule-based directed movement of a subset of apical surface proteins.

Live-cell imaging of the exocyst subunit Sec8 reveals how the protein’s spatiotemporal dynamics correlate with its roles in vesicle fusion., Tethers play ubiquitous roles in membrane trafficking and influence the specificity of vesicle attachment. Unlike soluble N-ethyl-maleimide–sensitive fusion attachment protein receptors (SNAREs), the spatiotemporal dynamics of tethers relative to vesicle fusion are poorly characterized. The most extensively studied tethering complex is the exocyst, which spatially targets vesicles to sites on the plasma membrane. By using a mammalian genetic replacement strategy, we were able to assemble fluorescently tagged Sec8 into the exocyst complex, which was shown to be functional by biochemical, trafficking, and morphological criteria. Ultrasensitive live-cell imaging revealed that Sec8-TagRFP moved to the cell cortex on vesicles, which preferentially originated from the endocytic recycling compartment. Surprisingly, Sec8 remained with vesicles until full dilation of the fusion pore, supporting potential coupling with SNARE fusion machinery. Fluorescence recovery after photobleaching analysis of Sec8 at cell protrusions revealed that a significant fraction was immobile. Additionally, Sec8 dynamically repositioned to the site of membrane expansion, suggesting that it may respond to local cues during early cell polarization.

Insulin releases an intracellular brake and promotes fusion pore expansion to translocate GLUT4 vesicles, and switches vesicle trafficking between distinct exocytic circuits., Insulin stimulates translocation of GLUT4 storage vesicles (GSVs) to the surface of adipocytes, but precisely where insulin acts is controversial. Here we quantify the size, dynamics, and frequency of single vesicle exocytosis in 3T3-L1 adipocytes. We use a new GSV reporter, VAMP2-pHluorin, and bypass insulin signaling by disrupting the GLUT4-retention protein TUG. Remarkably, in unstimulated TUG-depleted cells, the exocytic rate is similar to that in insulin-stimulated control cells. In TUG-depleted cells, insulin triggers a transient, twofold burst of exocytosis. Surprisingly, insulin promotes fusion pore expansion, blocked by acute perturbation of phospholipase D, which reflects both properties intrinsic to the mobilized vesicles and a novel regulatory site at the fusion pore itself. Prolonged stimulation causes cargo to switch from ∼60 nm GSVs to larger exocytic vesicles characteristic of endosomes. Our results support a model whereby insulin promotes exocytic flux primarily by releasing an intracellular brake, but also by accelerating plasma membrane fusion and switching vesicle traffic between two distinct circuits.

Although epithelial cells are known to exhibit a polarized distribution of membrane components, the pathways responsible for delivering membrane proteins to their appropriate domains remain unclear. Using an optimized approach to three-dimensional live cell imaging, we have visualized the transport of newly synthesized apical and basolateral membrane proteins in fully polarized filter-grown Madin–Darby canine kidney cells. We performed a detailed quantitative kinetic analysis of trans-Golgi network (TGN) exit, passage through transport intermediates, and arrival at the plasma membrane using cyan/yellow fluorescent protein–tagged glycosylphosphatidylinositol-anchored protein and vesicular stomatitis virus glycoprotein as apical and basolateral reporters, respectively. For both pathways, exit from the TGN was rate limiting. Furthermore, apical and basolateral proteins were targeted directly to their respective membranes, resolving current confusion as to whether sorting occurs on the secretory pathway or only after endocytosis. However, a transcytotic protein did reach the apical surface after a prior appearance basolaterally. Finally, newly synthesized proteins appeared to be delivered to the entire lateral or apical surface, suggesting—contrary to expectations—that there is not a restricted site for vesicle docking or fusion adjacent to the junctional complex.

Although cell movement is driven by actin, polarization and directional locomotion require an intact microtubule cytoskeleton that influences polarization by modulating substrate adhesion via specific targeting interactions with adhesion complexes. The fidelity of adhesion site targeting is precise; using total internal reflection fluorescence microscopy (TIRFM), we now show microtubule ends (visualized by incorporation of GFP tubulin) are within 50 nm of the substrate when polymerizing toward the cell periphery, but not when shrinking from it. Multiple microtubules sometimes followed similar tracks, suggesting guidance along a common cytoskeletal element. Use of TIRFM with GFP- or DsRed-zyxin in combination with either GFP-tubulin or GFP–CLIP-170 further revealed that the polymerizing microtubule plus ends that tracked close to the dorsal surface consistently targeted substrate adhesion complexes. This supports a central role for the microtubule tip complex in the guidance of microtubules into adhesion foci, and provides evidence for an intimate cross-talk between microtubule tips and substrate adhesions in the range of molecular dimensions.

Monitoring the fusion of constitutive traffic with the plasma membrane has remained largely elusive. Ideally, fusion would be monitored with high spatial and temporal resolution. Recently, total internal reflection (TIR) microscopy was used to study regulated exocytosis of fluorescently labeled chromaffin granules. In this technique, only the bottom cellular surface is illuminated by an exponentially decaying evanescent wave of light. We have used a prism type TIR setup with a penetration depth of ∼50 nm to monitor constitutive fusion of vesicular stomatitis virus glycoprotein tagged with the yellow fluorescent protein. Fusion of single transport containers (TCs) was clearly observed and gave a distinct analytical signature. TCs approached the membrane, appeared to dock, and later rapidly fuse, releasing a bright fluorescent cloud into the membrane. Observation and analysis provided insight about their dynamics, kinetics, and position before and during fusion. Combining TIR and wide-field microscopy allowed us to follow constitutive cargo from the Golgi complex to the cell surface. Our observations include the following: (1) local restrained movement of TCs near the membrane before fusion; (2) apparent anchoring near the cell surface; (3) heterogeneously sized TCs fused either completely; or (4) occasionally larger tubular-vesicular TCs partially fused at their tips.

Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcepsilonRI, is phosphorylated by the Src family tyrosine kinase Lyn to initiate mast cell signaling, leading to degranulation. Using fluorescence correlation spectroscopy (FCS), we observe stimulation-dependent associations between fluorescently labeled IgE-FcepsilonRI and Lyn-EGFP on individual cells. We also simultaneously measure temporal variations in the lateral diffusion of these proteins. Antigen-stimulated interactions between these proteins detected subsequent to the initiation of receptor phosphorylation exhibit time-dependent changes, suggesting multiple associations between FcepsilonRI and Lyn-EGFP. During this period, we also observe a persistent decrease in Lyn-EGFP lateral diffusion that is dependent on Src family kinase activity. These stimulated interactions are not observed between FcepsilonRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcepsilonRI implicated in downstream signaling events. They demonstrate the capacity of FCS cross-correlation analysis to investigate the mechanism of signaling-dependent protein-protein interactions in intact, living cells.