Cargo navigation across 3D microtubule intersections.

The eukaryotic cell's microtubule cytoskeleton is a complex 3D filament network. Microtubules cross at a wide variety of separation distances and angles. Prior studies in vivo and in vitro suggest that cargo transport is affected by intersection geometry. However, geometric complexity is not yet widely appreciated as a regulatory factor in its own right, and mechanisms that underlie this mode of regulation are not well understood. We have used our recently reported 3D microtubule manipulation system to build filament crossings de novo in a purified in vitro environment and used them to assay kinesin-1-driven model cargo navigation. We found that 3D microtubule network geometry indeed significantly influences cargo routing, and in particular that it is possible to bias a cargo to pass or switch just by changing either filament spacing or angle. Furthermore, we captured our experimental results in a model which accounts for full 3D geometry, stochastic motion of the cargo and associated motors, as well as motor force production and force-dependent behavior. We used a combination of experimental and theoretical analysis to establish the detailed mechanisms underlying cargo navigation at microtubule crossings.

Brain Slice Staining and Preparation for Three-Dimensional Super-Resolution Microscopy.

Localization microscopy techniques-such as photoactivation localization microscopy (PALM), fluorescent PALM (FPALM), ground state depletion (GSD), and stochastic optical reconstruction microscopy (STORM)-provide the highest precision for single-molecule localization currently available. However, localization microscopy has been largely limited to cell cultures due to the difficulties that arise in imaging thicker tissue sections. Sample fixation and antibody staining, background fluorescence, fluorophore photoinstability, light scattering in thick sections, and sample movement create significant challenges for imaging intact tissue. We have developed a sample preparation and image acquisition protocol to address these challenges in rat brain slices. The sample preparation combined multiple fixation steps, saponin permeabilization, and tissue clarification. Together, these preserve intracellular structures, promote antibody penetration, reduce background fluorescence and light scattering, and allow acquisition of images deep in a 30 µm thick slice. Image acquisition challenges were resolved by overlaying samples with a permeable agarose pad and custom-built stainless-steel imaging adapter, and sealing the imaging chamber. This approach kept slices flat, immobile, bathed in imaging buffer, and prevented buffer oxidation during imaging. Using this protocol, we consistently obtained single-molecule localizations of synaptic vesicle and active zone proteins in three dimensions within individual synaptic terminals of the striatum in rat brain slices. These techniques may be easily adapted to the preparation and imaging of other tissues, substantially broadening the application of super-resolution imaging.

CD169-mediated trafficking of HIV to plasma membrane invaginations in dendritic cells attenuates efficacy of anti-gp120 broadly neutralizing antibodies.

Myeloid dendritic cells (DCs) can capture HIV-1 via the receptor CD169/Siglec-1 that binds to the ganglioside, GM3, in the virus particle membrane. In turn, HIV-1 particles captured by CD169, an I-type lectin, whose expression on DCs is enhanced upon maturation with LPS, are protected from degradation in CD169+ virus-containing compartments (VCCs) and disseminated to CD4⁺ T cells, a mechanism of DC-mediated HIV-1 trans-infection. In this study, we describe the mechanism of VCC formation and its role in immune evasion mechanisms of HIV-1. We find HIV-1-induced formation of VCCs is restricted to myeloid cells, and that the cytoplasmic tail of CD169 is dispensable for HIV-1 trafficking and retention within VCCs and subsequent trans-infection to CD4⁺ T cells. Interestingly, introduction of a di-aromatic endocytic motif in the cytoplasmic tail of CD169 that results in endocytosis of HIV-1 particles, suppressed CD169-mediated HIV-1 trans-infection. Furthermore, super-resolution microscopy revealed close association of CD169 and HIV-1 particles in surface-accessible but deep plasma membrane invaginations. Intriguingly, HIV-1 particles in deep VCCs were inefficiently accessed by anti-gp120 broadly neutralizing antibodies, VRC01 and NIH45-46 G54W, and thus were less susceptible to neutralization. Our study suggests that HIV-1 capture by CD169 can provide virus evasion from both innate (phagocytosis) and adaptive immune responses.

NECAP 1 regulates AP-2 interactions to control vesicle size, number, and cargo during clathrin-mediated endocytosis.

AP-2 is the core-organizing element in clathrin-mediated endocytosis. During the formation of clathrin-coated vesicles, clathrin and endocytic accessory proteins interact with AP-2 in a temporally and spatially controlled manner, yet it remains elusive as to how these interactions are regulated. Here, we demonstrate that the endocytic protein NECAP 1, which binds to the α-ear of AP-2 through a C-terminal WxxF motif, uses an N-terminal PH-like domain to compete with clathrin for access to the AP-2 ß2-linker, revealing a means to allow AP-2-mediated coordination of accessory protein recruitment and clathrin polymerization at sites of vesicle formation. Knockdown and functional rescue studies demonstrate that through these interactions, NECAP 1 and AP-2 cooperate to increase the probability of clathrin-coated vesicle formation and to control the number, size, and cargo content of the vesicles. Together, our data demonstrate that NECAP 1 modulates the AP-2 interactome and reveal a new layer of organizational control within the endocytic machinery.

Asymmetric packaging of polymerases within vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) is a prototypic negative sense single-stranded RNA virus. The bullet-shape appearance of the virion results from tightly wound helical turns of the nucleoprotein encapsidated RNA template (N-RNA) around a central cavity. Transcription and replication require polymerase complexes, which include a catalytic subunit L and a template-binding subunit P. L and P are inferred to be in the cavity, however lacking direct observation, their exact position has remained unclear. Using super-resolution fluorescence imaging and atomic force microscopy (AFM) on single VSV virions, we show that L and P are packaged asymmetrically towards the blunt end of the virus. The number of L and P proteins varies between individual virions and they occupy 57 ± 12 nm of the 150 nm central cavity of the virus. Our finding positions the polymerases at the opposite end of the genome with respect to the only transcriptional promoter.

Actin mediates the nanoscale membrane organization of the clustered membrane protein influenza hemagglutinin.

The influenza viral membrane protein hemagglutinin (HA) is required at high concentrations on virion and host-cell membranes for infectivity. Because the role of actin in membrane organization is not completely understood, we quantified the relationship between HA and host-cell actin at the nanoscale. Results obtained using superresolution fluorescence photoactivation localization microscopy (FPALM) in nonpolarized cells show that HA clusters colocalize with actin-rich membrane regions (ARMRs). Individual molecular trajectories in live cells indicate restricted HA mobility in ARMRs, and actin disruption caused specific changes to HA clustering. Surprisingly, the actin-binding protein cofilin was excluded from some regions within several hundred nanometers of HA clusters, suggesting that HA clusters or adjacent proteins within the same clusters influence local actin structure. Thus, with the use of imaging, we demonstrate a dynamic relationship between glycoprotein membrane organization and the actin cytoskeleton at the nanoscale.

Superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells.

Localization-based superresolution optical imaging is rapidly gaining popularity, yet limited availability of genetically encoded photoactivatable fluorescent probes with distinct emission spectra impedes simultaneous visualization of multiple molecular species in living cells. We introduce PAmKate, a monomeric photoactivatable far-red fluorescent protein, which facilitates simultaneous imaging of three photoactivatable proteins in mammalian cells using fluorescence photoactivation localization microscopy (FPALM). Successful probe identification was achieved by measuring the fluorescence emission intensity in two distinct spectral channels spanning only ~100 nm of the visible spectrum. Raft-, non-raft-, and cytoskeleton-associated proteins were simultaneously imaged in both live and fixed fibroblasts coexpressing Dendra2-hemagglutinin, PAmKate-transferrin receptor, and PAmCherry1-ß-actin fusion constructs, revealing correlations between the membrane proteins and membrane-associated actin structures.

Nanoscale imaging of molecular positions and anisotropies.

Knowledge of the orientation of molecules within biological structures is crucial to understanding the mechanisms of cell function. We present a method to image simultaneously the positions and fluorescence anisotropies of large numbers of single molecules with nanometer lateral resolution within a sample. Based on a simple modification of fluorescence photoactivation localization microscopy (FPALM), polarization (P)-FPALM does not compromise speed or sensitivity. We show results for mouse fibroblasts expressing Dendra2-actin or Dendra2-hemagglutinin.

Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories.

Organization in biological membranes spans many orders of magnitude in length scale, but limited resolution in far-field light microscopy has impeded distinction between numerous biomembrane models. One canonical example of a heterogeneously distributed membrane protein is hemagglutinin (HA) from influenza virus, which is associated with controversial cholesterol-rich lipid rafts. Using fluorescence photoactivation localization microscopy, we are able to image distributions of tens of thousands of HA molecules with subdiffraction resolution ( approximately 40 nm) in live and fixed fibroblasts. HA molecules form irregular clusters on length scales from approximately 40 nm up to many micrometers, consistent with results from electron microscopy. In live cells, the dynamics of HA molecules within clusters is observed and quantified to determine an effective diffusion coefficient. The results are interpreted in terms of several established models of biological membranes.

Shape analysis of giant vesicles with fluid phase coexistence by laser scanning microscopy to determine curvature, bending elasticity, and line tension.

Membrane shape parameters such as curvature, bending elasticity, and lateral tension, are relevant to the lateral organization and function of biomembranes, and may critically influence the formation of lateral clustering patterns observed in living cells. Fluorescence laser-scanning microscopy can be used to image vesicles and cell membranes, and from shape analysis of these images mechanical membrane parameters can be quantified. Methods to analyze images of equatorial sections obtained by confocal or multiphoton microscopy are detailed, in order to estimate curvature, lateral tension, line tension, relative differences in mean curvature and Gaussian curvature bending moduli, and fluorescence dye intensity profiles, typically within coexisting liquid-ordered and liquid-disordered membrane domains. A variety of shape tracing and shape fitting methods are compared.

Imaging and shape analysis of GUVs as model plasma membranes: effect of trans DOPC on membrane properties.

Unsaturated trans fatty acids have been linked to a higher incidence of coronary artery disease, but not enough is known about the effect of trans lipids on membrane properties. Liquid-ordered (l(o)) and liquid-disordered (l(d)) membrane domains are implicated in various biological processes, such as endocytosis, adhesion, signaling, protein transport, apoptosis, and disease pathogenesis. The physical forces that induce domain formation and thus orchestrate cell function need to be further addressed and quantified. Here, we test the effect of trans DOPC (dielaidoyl phosphatidylcholine or DEPC) on the morphology of giant unilamellar vesicles (GUVs, used as a biomembrane model) made by electroformation with varying compositions of egg sphingomyelin, trans DOPC, cis DOPC, and cholesterol. GUVs were imaged by confocal fluorescence microscopy and then analyzed for changes in membrane morphology and properties such as l(o)/l(d) phase coexistence and area fractions, distribution of meridional curvature, and fluorescent-probe intensity distribution. BODIPY-FL-C(12)-sphingomyelin, Lissamine rhodamine B dioleoylphosphatidylethanolamine and BODIPY-TR-C(12)-sphingomyelin were used as fluorescent probes to differentially label the l(o) and l(d) phases. Trans DOPC induces some vesicles to form multidomain, invaginated morphologies that differ from the typical two-domain circular and truncated spherical shapes observed in its absence. Trans DOPC also alters the membrane curvature distribution; this is more pronounced in the l(o) phase near the phase boundary, where significantly negative curvatures (<-0.5 microm(-1)) are observed. A narrower distribution of meridional curvatures in GUVs with trans DOPC is suggestive of higher membrane bending rigidity. The ratio of average fluorescent intensities in the l(d)/l(o) phases indicates a greater concentration or brightness of the probes BODIPY-FL-C(12)-sphingomyelin and BODIPY-TR-C(12)-sphingomyelin in the l(o) phase in the presence of trans DOPC. Addition of trans DOPC does not alter the l(o)/l(d) area fractions, indicating that it does not act like egg sphingomyelin, a saturated lipid. These changes in membrane properties seen in the presence of trans lipids could significantly impact cell function.

Combined interaction of phospholipase C and apolipoprotein A-I with small unilamellar lecithin-cholesterol vesicles: influence of apolipoprotein A-I concentration and vesicle composition.

We report the combined effects of phospholipase C (PLC), a pronucleating factor, and apolipoprotein A-I (apo A-I), an antinucleating factor, in solutions of model bile. Results indicate that apo A-I inhibits cholesterol nucleation from unilamellar lecithin vesicles by two mechanisms. Initially, inhibition is achieved by apo A-I shielding of hydrophobic diacylglycerol (DAG) moieties so as to prevent vesicle aggregation. Protection via shielding is temporary. It is lost when the DAG/apo A-I molar ratio exceeds a critical value. Subsequently, apo A-I forms small ( approximately 5-15 nm) complexes with lecithin and cholesterol that coexist with lipid-stabilized (400-800 nm) DAG oil droplets. This microstructural transition from vesicles to complexes avoids nucleation of cholesterol crystals and is a newly discovered mechanism by which apo A-I serves as an antinucleating agent in bile. The critical value at which a microstructural transition occurs depends on binding of apo A-I and so varies with the cholesterol mole fraction of vesicles. Aggregation of small, unilamellar, egg lecithin vesicles (SUVs) with varying cholesterol composition (0-60 mol %) was monitored for a range of apo A-I concentrations (2 to 89 microg/mL). Suppression of aggregation persists so long as the DAG-to-bound-apo A-I molar ratio is less than 100. A fluorescence assay involving dansylated lecithin shows that the suppression is an indirect effect of apo A-I rather than a direct inhibition of PLC enzyme activity. The DAG-to-total apo A-I molar ratio at which suppression is lost increases with cholesterol because of differences in apo A-I binding. Above this value, a microstructural transition to DAG droplets and lecithin/cholesterol A-I complexes occurs, as evidenced by sudden increases in turbidity and size and enhancement of Forster resonance energy transfer; structures are confirmed by cryo TEM.

Interaction of apolipoprotein A-I with lecithin-cholesterol vesicles in the presence of phospholipase C.

Here we study the anti-nucleating mechanism of apolipoprotein A-I (apo A-I) on model biliary vesicles in the presence of phospholipase C (PLC) utilizing dynamic light scattering (DLS), steady-state fluorescence spectroscopy, cryogenic transmission electron microscopy (cryo-TEM), and UV/Vis spectroscopy. PLC induces aggregation of cholesterol-free lecithin vesicles from an initial, average size of 100 nm to a maximal size of 600 nm. The presence of apo A-I likely inhibits vesicle aggregation by shielding the PLC-generated hydrophobic moieties, which results in vesicles of an average size of 200 nm. A similar phenomenon is observed in cholesterol-enriched lecithin vesicles. Whereas PLC alone produces aggregates of 300 nm, no aggregation is observed when apo A-I is present along with PLC. However, the ability of apo A-I to inhibit aggregation is temporary, and after 8 h, a broad particle size distribution with sizes as high as 800 nm is observed. Apo A-I possibly induces the formation of small apo A-I/lecithin/cholesterol complexes of about 5-20 nm similar to the discoidal pre-HDL complexes found in blood when it can no longer effectively shield all the DAG molecules. Concomitant with formation of complexes, DAG molecules coalesce into large oil droplets, which account for the large particles observed by light scattering. Thus, apo A-I acts as an anti-nucleating agent by two mechanisms, anti-aggregation and microstructural transition. The mode of protection is dependent on the cholesterol content and the relative amounts of DAG and apo A-I present. This study supports the possibility of apo A-I solubilizing lipids in bile in a similar fashion as it does in blood and also delineates the mechanism of formation of the complexes.