Nup358 associates with HIV-1 cores in the cytoplasm during infection.

<p><b>(A)</b> MDM or HeLa cells were synchronously infected HIV-1 GFP pseudotyped with VSV-g bearing either the wildtype (WT) CA or N74D and P90A CA mutants(MDMs MOI 0.3, HeLa MOI 0.6).Cells fixed at 3h post infection, incubated with primary antibodies to Nup358 and HIV-1 CA, followed by secondary antibodies specific for these antibodies conjugated to the PLUS and MINUS PLA oligonucleotides. Each red fluorescent puncta represents a positive PLA signal generated by the interaction of PLUS and MINUS oligonucleotides bound to secondary antibodies. <b>(B)</b> Quantification of PLA signal in MDMs and HeLa cells, as measured by the average fold increase in PLA signal, relative to uninfected control, in three independent experiments.<b>(C)</b> HeLa cells were transfected with siRNA’s targeting KIF5B, Nup358 or scrambled control siRNA and synchronously infected with R7ΔEnvGFPpseudotyped with VSV-g (MOI 0.6) 96 hours following siRNA transfection. Cells fixed at 3h post infection and PLA assay performed as described in (A). <b>(D)</b> Quantification of PLA signal in the siRNA knockdown cells as in (B). 20 or more cells were analyzed in each experiment. Error bars represent the SEM(**p<0.01, *p<0.05, ns = not significant). Data is representative of three or more independent experiments.</p

HIV-1 capsid influences the dependence on KIF5B and Nup358 mediated nuclear import during infection.

<p>HeLa cells treated with scrambled, Nup358 or KIF5B siRNA for 72h were subjected to synchronized infected with 100ng of VSVg pseudotyped HIV-1 reporter virus bearing either the wildtype (WT) CA or N74D and P90A CA mutants. Cells were collected 24 post infection and real time PCR performed using specific primers to quantify Late RT <b>(A)</b> and 2-LTR circle <b>(B)</b>. Error bars represent the standard deviation of single samples run in triplicate. <b>(C)</b> GFP expression 48h post infection, as measured by flow cytometry of 10,000 cells. The data shown here is representative of three independent experiments.</p

HIV-1 infection induces Nup358 relocalization.

<p><b>(A,B)</b> Monocyte derived macrophages (MDM) and HeLa cells were synchronously infected with VSVg pseudotyped HIV-1 reporter virus (MOI 0.3 for MDM and MOI 0.6 for HeLa cells) bearing either the wildtype (WT) CA or N74D and P90A CA mutants. Cells were fixed at 0, 1 or 3h (shown) post infection and stained for Nup358 (green). Infection for each cell type is shown <b>(B)</b>The fraction of Nup358 signal in the cytoplasm at the indicated time PI, measured as in 2C.<b>(C,D)</b>TZM-Bl cells were synchronously infected with R7ΔEnvGFPpseudotyped with the HXB2 envelope protein (MOI 0.32). Cells were fixed 0, 1 and 3h (shown) post infection and stained for Nup358. <b>(D)</b> The fraction of Nup358 signal in the cytoplasm at the indicated time PI.<b>(E,F)</b>HeLa cells were transfected with KIF5B specific or scrambled control siRNA and synchronously infected with R7ΔEnvGFPpseudotyped with VSV-g (MOI 0.6)96 hours following siRNA transfection. Cells were fixed 0, 1 and 3h (shown) post infection and stained for Nup358.<b>(F)</b>The fraction of Nup358 signal in the cytoplasm at the indicated time PI.20 or more cells were analyzed for each sample. Error bars represent the SEM of three independent experiments. (**p<0.01, *p<0.05, ns = not significant). Data is representative of three or more independent experiments.</p

KIF5B and NUP358 facilitate HIV-1 uncoating.

<p><b>(A)</b> HeLa cells were transfected with siRNA’s targeting Nup358, KIF5B or both (Nup358+KIF5B). Expression of the indicated proteins were determined by western blot 96h post transfection.<b>(B)</b> siRNA treated HeLa cells infected with VSVg-HIV-1 GFP (MOI 0.8) and infectivity measured by determining the percent of GFP positive cells by FACS. <b>(C)</b> HeLa cells treated with scrambled, Nup358, KIF5B or Nup358+5B siRNA for 96h were synchronously infected with S15-mCherry/GFP-Vpr VSVg-HIV-1. Cells were fixed at the indicated time point, and the p24 intensities associated with individual virions lacking the S15 membrane label (1–3 hours PI) or all virions (controls and 0 hr points) are shown. Bafilomycin A1 (Baf1), inhibits VSVg mediated fusion and was used as a control in these experiments. Red line is the average p24 intensity measured for all fused viruses at the indicated time point. At least 20 cells were imaged at each time point. Error bars represent SEM.<b>(D)</b>Data from three independent experiments, as shown in C, were normalized to the mean p24 intensity observed in control siRNA transfected cell and averaged. **p<0.01, *p<0.05, ns = not significant. Data is representative of three or more independent experiments.</p

Nup358 association with HIV-1 cores is CPSF6 dependent and Cyclophilin A independent.

<p>HeLa cells transfected with scrambled siRNA or siRNA specific for CPSF6 or CypA.<b>(A)</b>Western blot for CPSF6 or CypA 96h following siRNA transfection. <b>(B)</b>siRNA depleted cells were synchronously infected with VSVg-R7ΔEnvGFP(MOI 0.6). Cells were fixed 0, 1 and 3h post infection stained for HIV-1 capsid protein p24 (red) and NUP358 (green).Depicted a representative image at 3h post infection. Nup358 and CA colocalization following CypA depletion shown in bottom panel. <b>(C)</b>The fraction of Nup358 signal in the cytoplasm at the indicated time PI. <b>(D)</b> Quantification of CA and Nup358 signal colocalization in the siRNA depleted cells. <b>(E)</b> PLA of Nup358 and CA, performed on the siRNA depleted cells fixed 3 hours following synchronous infection, and quantification of average fold increase in PLA signal. 20 or more cells were analyzed in each experiment. Error bars represent the SEM (**p<0.01, *p<0.05, ns = not significant). Data is representative of three or more independent experiments.</p

KIF5Band Nup358 depletion lead to the perinuclear accumulation of HIV-1 cores.

<p>HeLa cells were transfected with siRNA’s targeting Nup358, KIF5B or a scrambled siRNA sequence.<b>(A)</b> Western blot for KIF5B or NUP358 96h following siRNA transfection <b>(B)</b>siRNA depleted cells were synchronously infected withVSVg-R7ΔEnvGFP(MOI 0.6). Cells were fixed 0, 1and 3h following a synchronized infection and stained for HIV-1 capsid protein p24 (red) and DAPI (blue) for cell nuclei. A representative image at 3h post infection is depicted.<b>(C)</b>Quantification process employed to detect perinuclear and cytoplasmic p24 protein levels. Nuclear mask generated based on the DAPI channel (left image) and perinuclear signal quantified by masking all signal inside (middle image) or outside right image) of the nuclear mask. <b>(D)</b>Percentage ofp24 puncta in the perinuclear region in KIF5B and Nup358 depleted cells, quantified as described in (C). TZM-bl cells were transfected with siRNA’s targeting Nup358, KIF5B or a scrambled siRNA sequence.<b>(E)</b> Western blot for KIF5B or NUP358 72h following siRNA transfection. siRNA depleted cells were synchronously infected with HXB2-R7ΔEnvGFP(MOI 0.32). <b>(F)</b>Infectivity was assessed 48 hours following infection. <b>(G,H)</b> Cells were fixed 0, 1and 3h following a synchronized infection and stained for HIV-1 capsid protein p24 (red) and DAPI (blue) for cell nuclei. A representative image at 3h post infection is depicted. <b>(H)</b> Percentage ofp24 puncta in the perinuclear region in KIF5B and Nup358 depleted cells, quantified as described in (C). 20 or more cells was analyzed at each time point. Error bars represent the SEM of three experiments. The total number of virions analyzed at each time point is shown below each graph. ***p<0.001, *p<0.05, ns = not significant. Data is representative of three or more independent experiments.</p

Anterograde trafficking of HIV-1 cores during infection.

<p>HeLa cells transfected with a NUP358-GFP expressing BAF were synchronously infected with GIR labelled HIV-1 viral particles (MOI 0.3). 2 hours after infection, cells were imaged every 15 seconds for 10 minutes. Shown are individual z-planes showing a virus observed to traffic away from the nucleus during the acquisition period while associated with NUP358-GFP.</p

Primer Sequences.

<p>Primer Sequences.</p

Flow cytometric analysis of primary HUCs.

<p>Cells were stained with eight fluorochrome-conjugated monoclonal antibodies direct against cell surface makers. Cells were analyzed for the expression of: (A) HLA-ABC and CD54 (ICAM-1); (B) CD104 and EpCAM; (C) HLA-DR, and CD44– expression of CD44 shows two distinct populations of HUCs: CD44^lo and CD44^hi; (D, E) TLR and CD14. Representative data from 3 experiments performed with HUCs generated from 3 independent bladder biopsies.</p

Alpha-Synuclein Induces Lysosomal Rupture and Cathepsin Dependent Reactive Oxygen Species Following Endocytosis

<div><p>α-synuclein dysregulation is a critical aspect of Parkinson's disease pathology. Recent studies have observed that α-synuclein aggregates are cytotoxic to cells in culture and that this toxicity can be spread between cells. However, the molecular mechanisms governing this cytotoxicity and spread are poorly characterized. Recent studies of viruses and bacteria, which achieve their cytoplasmic entry by rupturing intracellular vesicles, have utilized the redistribution of galectin proteins as a tool to measure vesicle rupture by these organisms. Using this approach, we demonstrate that α-synuclein aggregates can induce the rupture of lysosomes following their endocytosis in neuronal cell lines. This rupture can be induced by the addition of α-synuclein aggregates directly into cells as well as by cell-to-cell transfer of α-synuclein. We also observe that lysosomal rupture by α-synuclein induces a cathepsin B dependent increase in reactive oxygen species (ROS) in target cells. Finally, we observe that α-synuclein aggregates can induce inflammasome activation in THP-1 cells. Lysosomal rupture is known to induce mitochondrial dysfunction and inflammation, both of which are well established aspects of Parkinson's disease, thus connecting these aspects of Parkinson's disease to the propagation of α-synuclein pathology in cells.</p></div