Explore the vast range of titles available.

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.

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.

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 ([almost equal to]40 nm) in live and fixed fibroblasts. HA molecules form irregular clusters on length scales from [almost equal to]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.

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 β2-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.

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 μm −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.

A simple modification to the optical configuration used for fluorescence photoactivation localization microscopy (FPALM) allows the fluorescence anisotropies of each individual molecule in a nanoscale image to be measured. The method was used to obtain position and orientation information for fluorescently labeled actin or hemagglutinin molecules in fixed fibroblasts. 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.

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.

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 β2-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.

Gallstone disease is the most common gastrointestinal disease that afflicts 10–15% of the US population with an annual cost of over $10.5 billion. Though the aetiolgy of the disease is well understood, more research is warranted regarding the pathogenesis. Knowledge about the gallstone pathway will aid in the measures for prophylactic management. Gallstones form due to the precipitation of cholesterol from cholesterol-supersaturated bile. Small unilamellar vesicles (SUVs) are the predominant transporters of cholesterol in bile. Precipitation of cholesterol monohydrate crystals from aggregated and/or fused SUVs in cholesterol-supersaturated bile followed by crystal accretion and growth leads to gallstone formation. The rate at which cholesterol nucleates from the vesicles, which is influenced by a host of biliary factors, plays a pivotal role in determining the lithogenicity of bile. The kinetic factors are divided into pro-nucleating and anti-nucleating factors that enhance and inhibit nucleation respectively. The balance between the two factors is largely responsible for the incidence of gallstones. This research focuses on understanding the concerted influence of phospholipase C (PLC), a pro-nucleating enzyme, and apolipoprotein (apo A-I), an anti-nucleating protein, on small unilamellar vesicles. We find that apo A-I exerts its anti-nucleating protection via two mechanisms, anti-aggregation and complex formation. The mode of protection is dependent on the vesicle composition, and PLC and apo A-I concentrations. It has been previously speculated that apo A-I/lipid complexes exist in bile and this research effort provides proof and confirms prior allusions. The similarities in physical chemistry between gallstone disease and atherosclerosis enable this research to be applied in the study of coronary heart disease.