Fig 1: Leishmania degrades the NPC of macrophages in a GP63-dependent manner.(A) L. major degrades NPC Nups. LM1 MΦ cells were infected as indicated with L. major for different times. Cell lysates were submitted to total protein lysis. Parasite lysate (Lmaj Lys) was used as a control. Samples were analyzed either by western blot or by gelatin zymography. An antibody against Leishmania GP63 was used to monitor its presence. An antibody against FG-Nups was used to monitor NPC degradation. In addition, antibodies against the FG-Nups Nup358, Nup214, Nup62, and against Nup93 were used. (Also see S1B Fig) (B) NPC degradation is GP63-dependent. LM1 MΦ cells were infected as indicated with either L. mexicana or L. major species (WT, GP63-/-, GP63R) for 2 hrs. Total protein lysates were analyzed by western blot. Parasite lysate (Lmaj Lys) was used as a control. A FG-Nup antibody was used to monitor the NPC degradation and a GP63 antibody to monitor its presence. (C) GP63 does not need the NLS but needs to be active to degrade the NPC. LM1 MΦ cells were infected as indicated with L. major (WT, GP63-/-, GP63R, GP63NLS, GP63AS) for 2 hrs. Total protein lysates were analyzed by western blot. Antibodies against Nup358 and Nup62 were used to monitor the NPC degradation and a GP63 antibody to monitor its presence. (D) Confirmation of GP63-dependent Nup62 degradation by confocal microscopy. LM1 MΦ cells were infected as indicated with different species of L. major (WT, GP63-/-, GP63R) for 2 hrs. Cells were stained for Nup62. White arrows represent Nup62, green arrows represent parasites. Results are representative of 3 sets of experiments. (E) Quantification of Nup62 after infection with different species of L. major (WT, GP63-/-) in whole nuclei (WN), nuclear envelope (NE), and nucleoplasm (NP). Values are represented +/- SEM. *** p < 1.10–3.
Fig 2: Structural organization and interactions of BICD2, RanBP2, and Nesprin-2.(A) Schematic representation of BicD2 (top), RanBP2 (middle), and Nesprin-2 (bottom). BicD2 (top): The colored boxes represent the three coiled-coil domains (CC1, CC2, and CC3) of the BICD2 protein. Interactors for each domain are noted on top. RanBP2 (Middle): A drawing of RanBP2 (modified from [14]), showing leucine-rich region (LRR), an E3 Sumo ligase domain (E3), and four Ran-binding domains (R), two of which flanking the BicD2 binding domain (BBD). “BBD” (highlighted in blue) of RanBP2 is used in the biochemical assays of this study. Nesprin-2 (bottom): A drawing of Nesprin-2 [3, 21] with “N2G 52–56” highlighted in green. The BicD2 binding “N2G 52–56” fragment [3] is used in the biochemical assays of this study. (B) Schematic depiction of BICD2 in an autoinhibited state. (C) Drawing of the cerebral cortex during development (Left). Both the apical nuclear migration of Radial Glial Progenitors and the basal migration of post-mitotic neurons are mediated by dynein, which is recruited to the nuclear envelope via two distinct pathways: (2) RanBP2- or (1) Nesprin-2-BICD2 interactions (Right). (D) Left panel: BICD2 fragments used in the GST pull-down (Top left). A Coomassie-stained SDS-PAGE of the purified BICD2 fragments used in the GST pull-down assay is shown below. Middle panel: An SDS-PAGE of the elution fractions of the GST-pulldown assay is shown. The GST-tagged RanBP2 fragment (BICD2 binding domain, aa 2148–2240) pulls down the BICD2 fragments aa 630–800 and aa 711–800, but not the fragment aa 750–800. The GST-tagged Nesprin-2 fragment (BICD2/Dynein binding domain, for the sequence see S1A Fig) pulls down each of the three BICD2 fragments. A pull-down assay with GST is shown as a negative control. The molar masses of standards are indicated on the left of each SDS-PAGE. Right panel: The intensities of the BICD2 gel bands from the pull-down assays with Nesprin-2 were quantified from 3 sets of experiments, a representative dataset is shown in S1B Fig. Discrete amounts of the three BICD2 fragments were analyzed on the SDS-PAGE and used as calibration standards to calculate the amounts of BICD2 bound to Nesprin-2 (μg) from the pull-down assays. Since the staining intensities are directly proportional to the molar masses of each fragment, in order to compare the number of bound BICD2 molecules, the BICD2 amounts were converted to the molar concentration divided by the molar concentration of Nesprin-2 and normalized to 1 for the longest fragment. The error was calculated as the standard deviation. Student’s t-test was performed against the BICD2 (aa 630–800) condition and the statistical significance is shown. (**** p<0.0001 and ns = not significant).
Fig 3: HIV-1 infection induces Nup358 relocalization. (A,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)The fraction of Nup358 signal in the cytoplasm at the indicated time PI, measured as in 2C.(C,D)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. (D) The fraction of Nup358 signal in the cytoplasm at the indicated time PI.(E,F)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.(F)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.
Fig 4: Anterograde trafficking of HIV-1 cores during infection.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.
Fig 5: Effects of depleting individual Nups on NPC integrity and function, and MX2 localization.Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoechst-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.
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