Journal: Virology Journal

Article Title: Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5

doi: 10.1186/1743-422X-11-40

Figure Lengend Snippet: Effect of dominant negative Rab GTPases on ABLV G-mediated viral entry. HEK293T cells expressing dsRed-tagged WT and DN Rab5, Rab7, and Rab11were infected with maxGFP encoding rVSV that express ABLVp G, ABLVs G, or VSV G at a MOI = 3 for 8 hrs or with rVSV that expresses EboGP at a MOI = 15 for 20 hrs and then analyzed as described in Figure . Infection of all cells was assessed. Under these conditions, the chosen MOIs yielded 40-50% virus-infected cells in controls. Results are expressed as percent virus-infected cells relative to that of WT Rab controls and represent 3 independent experiments; error bars are SEM. (A) Rab5. (B) Rab7. (C) Rab11. **, p < 0.0001; *, p < 0.005.

Article Snippet: DsRed-tagged Rab7 wild-type (WT) (plasmid #12661) and DN (T22N; plasmid #12662), DsRed-tagged Rab11 WT (plasmid #12679) and DN (S25N; plasmid #12680) [ ], and mRFP-tagged Rab5 WT (plasmid #14437) [ ] were purchased from Addgene, Cambridge, MA.

Techniques: Dominant Negative Mutation, Expressing, Infection, Virus

Journal: Journal of extracellular vesicles

Article Title: Breast Cancer-Derived Extracellular Vesicles Modulate the Cytoplasmic and Cytoskeletal Dynamics of Blood-Brain Barrier Endothelial Cells.

doi: 10.1002/jev2.70038

Figure Lengend Snippet: FIGURE 1 Extracellular vesicles derived from brain-seeking MDA-MB-231 cells (Br-EVs) modulate the expression of multiple Rab11 effector proteins (rab11fips) involved in intracytoplasmic vesicle trafficking and recycling. (a) Schematic representing degradation and recycling pathways in brain endothelial cells (BECs). Created using Biorender. (b) Schematic depicting the most widely acknowledged mechanisms of interaction between Rab11fip2, Rab11fip3, Rab11fip5, and the respective motor proteins for the tethering and transport of rab11 vesicles on cytoskeleton filaments. Created using Biorender. (c–e) Western blot quantification, representative immunofluorescent image, and fluorescence intensity quantification of Rab11fip2, (f–h) Rab11fip3, and (j–k) Rab11fip5 protein expression in BECs following treatment with PBS, P-EVs, and Br-EVs (mean ± SD; technical triplicates in 3

Article Snippet: The cell pellet was resuspended in 100 μL of buffer, mixed with 2 μg of Rab11fip3 (human tagged orf clone rc227246, Origene Technologies Inc.) and Rab11fip5 (human tagged orf clone rc206173, Origene Technologies Inc.) plasmids, and electroporated using the S-05 Program of the Amaxa Biosystem Nucleofector.

Techniques: Derivative Assay, Expressing, Western Blot, Fluorescence

Journal: Journal of extracellular vesicles

Article Title: Breast Cancer-Derived Extracellular Vesicles Modulate the Cytoplasmic and Cytoskeletal Dynamics of Blood-Brain Barrier Endothelial Cells.

doi: 10.1002/jev2.70038

Figure Lengend Snippet: FIGURE 2 Cerebral microvessels isolated from the brain cortex of mice treated with Br-EVs show decreased Rab11fip2 and increased Rab11fip3 and Rab11fip5 levels in response to Br-EV treatment. (a) Western blot quantification of Rab11fip2, Rab11fip3, and Rab11fip5 in cerebral microvessel lysates of mice treated with PBS, P-EVs, and Br-EVs. PBS, P-EV and Br-EV groups were run on separate gels under the same conditions and normalization to GAPDH was performed individually for each sample with raw values being presented. Uncropped gels are provided in the Figure S5 (mean ± SD; n = 4 for PBS and Br-EV groups and n = 6 and 5 for the P-EV group). (b) Representative fluorescent images of cerebral microvessels from the brains of mice treated with PBS, P-EVs, and Br-EVs and stained with Rab11fip2, Rab11fip3, and Rab11fip5 (green) and nuclei (blue). Scale bars 50 µm. (c) Quantification of the signal mean fluorescence intensity (MFI) of the cerebral microvessels of mice treated with PBS, P-EVs, and Br-EVs and stained for Rab11fip2, Rab11fip3, and Rab11fip5 (normalized to the image background; mean ± SD; n = 5 for the PBS group, n = 6 for the P-EV group, and n = 4 for the Br-EV group). Statistical analysis was performed using the Mann–Whitney test (a) and one-way ANOVA with Tukey’s multiple comparison test (c) (Wu and Voeltz 2021; Lee et al. 2019; Mohrmann et al. 2002; Busatto et al. 2020). ns = not significant; *p ≤0.0332; **p ≤0.0021; ****p ≤0.0001.

Article Snippet: The cell pellet was resuspended in 100 μL of buffer, mixed with 2 μg of Rab11fip3 (human tagged orf clone rc227246, Origene Technologies Inc.) and Rab11fip5 (human tagged orf clone rc206173, Origene Technologies Inc.) plasmids, and electroporated using the S-05 Program of the Amaxa Biosystem Nucleofector.

Techniques: Isolation, Western Blot, Staining, Fluorescence, MANN-WHITNEY, Comparison

Journal: Journal of extracellular vesicles

Article Title: Breast Cancer-Derived Extracellular Vesicles Modulate the Cytoplasmic and Cytoskeletal Dynamics of Blood-Brain Barrier Endothelial Cells.

doi: 10.1002/jev2.70038

Figure Lengend Snippet: FIGURE 5 BECs knocked down (KD) for Rab7 and overexpressing (OE) Rab11fip5 display motility and morphology patterns similar to those of BECs treated with Br-EVs. UMAP representation of BEC populations KD for Rab7 and Rab11fip2 and OE for Rab11fip3 and Rab11fip5 (from left to right), analysed for morphology features using (a) confocal microscopy (to be compared with Figure 3c) and (c) live cell imaging (to be compared with Figure 3j), and for motility features using (e) QPI (to be compared with Figure 4c) and (g) live cell imaging (to be compared with Figure 4j). (b, d, f and h) Tables showing variations in cluster distribution and the percentage of each cluster within each respective sample.

Article Snippet: The cell pellet was resuspended in 100 μL of buffer, mixed with 2 μg of Rab11fip3 (human tagged orf clone rc227246, Origene Technologies Inc.) and Rab11fip5 (human tagged orf clone rc206173, Origene Technologies Inc.) plasmids, and electroporated using the S-05 Program of the Amaxa Biosystem Nucleofector.

Techniques: Confocal Microscopy, Live Cell Imaging

Journal: Neuroscience Bulletin

Article Title: Myosin Va-dependent Transport of NMDA Receptors in Hippocampal Neurons

doi: 10.1007/s12264-023-01174-y

Figure Lengend Snippet: The interaction of MyoVa with FIP3 is required for NMDAR transport. A Schematic of MyoVa and its association with cargo proteins via Rab11 and FIPs. B Co-IP between FIP3 and associated proteins with antibodies to FIP3 in control or under PMA conditions. Non-immune IgG was used as control. C Statistical analysis of binding between FIP3 and associated proteins. Signals were divided by FIP3 signals and normalized to control. MyoVa: 1.33 ± 0.06, P <0.01; GluN1: 1.44 ± 0.06, P <0.01; GluN2A: 1.45 ± 0.05, P <0.01; Rab11: 1.38 ± 0.08, P <0.01; n = 9; unpaired Student’s t -test vs control. D PMA enhances the interaction of FIP3 and GluN1, revealed by co-IP between FIP3 and GluN1 with antibodies to GluN1 in control or under PMA conditions. Non-immune IgG was used as control. E Statistical analysis of binding between FIP3 and GluN1. FIP3 signals were divided by GluN1 signals and normalized to control. FIP3: 1.37 ± 0.12, P <0.05, n = 8; unpaired Student’s t -test vs control. F Representative images display increased colocalization of FIP3 and GluN1 in primary hippocampal neurons stained with GluN1 (green) and FIP3 (red) antibodies in control or under PMA conditions. Scale bar, 5 μm. G Quantification of the colocalization between GluN1 and FIP3 by Pearson’s coefficient. Control: n = 20 neurons, 0.62 ± 0.01; PMA: n = 26 neurons, 0.71 ± 0.01, P <0.01; data are from at least three independent cultures; unpaired Student’s t -test vs control. H Illustration of the locations used for unilateral viral injections into the hippocampal CA1 region. The virus expresses FIP3 KD or scrambled shRNA and is reported by EGFP (green). I–K FIP3 KD blocks the PMA-induced LTP of NMDA fEPSPs. The overlaid traces display changes in the average response selected at the times shown (marked by 1 and 2). J Cumulative probability of potentiation magnitude of NMDA fEPSPs. K Summary graphs of LTP magnitude from experiments shown in I . Control: n = 7, 1.69 ± 0.07; FIP3 KD: n = 8, 1.01 ± 0.01, P <0.01; scrambled: n = 7, 1.79 ± 0.09; P >0.05; one-way repeated-measures ANOVA vs control. Scale bars, 0.5 mV, 100 ms in I . L–N As in I–K , with the exception that LTP of NMDA fEPSPs was elicited by TBS. Control: n = 7, 1.69 ± 0.06; FIP3 KD: n = 7, 0.98 ± 0.02, P <0.01; scrambled: n = 7, 1.75 ± 0.08, P >0.05; one-way repeated-measures ANOVA vs control. Scale bars, 0.5 mV, 100 ms in L . The data are represented as the mean ± SEM, * P <0.05; ** P <0.01. ns, no significant difference.

Article Snippet: The major primary antibodies were: MyoVa (Sigma, St. Louis, USA, HPA001356), GluN2A (Millipore, Burlington, USA, AB1555P), GluN1 (rabbit: Abclonal, Wuhan, China, A11699; mouse: Abcam, Cambridge, UK, ab134308), GluA1 (Millipore, MAB2263), GluN2B (Cell Signaling Technology, Boston, USA, 14544), Rab11 (Invitrogen, Carlsbad, USA, 71-5300), FIP3 (Rockland, Philadelphia, USA, 600-401-994), PSD95 (Millipore, MABN68), MyoVb (Sigma, HPA040902), CaMKII (Santa Cruz Biotechnology, Inc., Dallas, USA, sc-13141), GST (glutathione-S-transferase, Genscript, Nanjing, China, A00866-100).

Techniques: Co-Immunoprecipitation Assay, Control, Binding Assay, Staining, Virus, shRNA

Journal: Neuroscience Bulletin

Article Title: Myosin Va-dependent Transport of NMDA Receptors in Hippocampal Neurons

doi: 10.1007/s12264-023-01174-y

Figure Lengend Snippet: FIP3 KD impairs hippocampal memory. A Schematic showing the locations used for bilateral viral injections into the CA1 region of the hippocampus. B Representative images showing EGFP expression in the CA1 region of the dorsal hippocampus prepared from FIP3 KD rats. Scale bars, 250 μm and 75 μm (enlarged images). C, D FIP3 KD rats show deficits in contextual ( C ) and trace fear memory ( D ) tested at 24 h, whereas the scrambled animals showed freezing comparable to control rats. FD, fear conditioning. C : control, n = 14 rats, 62.46% ± 3.52%; FIP3 KD, n = 9 rats, 32.33% ± 5.13%, P <0.01; scrambled, n = 8 rats, 62.23% ± 5.56%; P >0.05; one-way repeated-measures ANOVA vs control. D : Control: n = 14 rats, tone, 58.82% ± 6.40%; trace 68.73% ± 6.40%; ITI, 56.60% ± 5.45%; FIP3 KD: n = 8 rats, tone, 32.76% ± 7.21%, P <0.05; trace, 38.96% ± 7.94%, P <0.05; ITI, 33.39% ± 7.72%, P <0.05; scrambled: n = 8 rats, tone, 59.90% ± 8.03%, P >0.05; trace, 71.62% ± 6.31%, P >0.05; ITI, 65.72% ± 6.51%, P >0.05; two-way repeated-measures ANOVA vs control. E Schematic of the novel object preference (NOR) task. F, G FIP3 KD rats show deficits in the NOR task. In the acquisition phase, the FIP3 KD, scrambled, and control rats spend similar amounts of time exploring the two objects. In the test phase tested at 24 h, control and scrambled rats prefer the novel objects, whereas the FIP3 KD rats exhibit a lower preference for the novel objects. Acquisition: control, n = 10 rats, 49.58% ± 1.43%; FIP3 KD, n = 12 rats, 49.34% ± 2.87%, P >0.05; scrambled, n = 10 rats, 51.09% ± 2.21%, P >0.05. Test: control, n = 10 rats, 65.62% ± 1.67%; FIP3 KD, n = 12 rats, 52.87% ± 3.92%, P <0.05; scrambled, n = 10 rats, 67.65% ± 3.70%, P >0.05; one-way repeated-measures ANOVA vs control. H Schematic of novel place preference test (NPP). I, J FIP3 KD rats show deficits in the NPP task. In the acquisition phase of the object location task, FIP3 KD, scrambled, and control rats spend similar time exploring the two objects. In the test phase at 24 h, control and scrambled rats prefer the relocated object, whereas the FIP3 KD rats have a lower preference for the relocated object. Acquisition: control, n = 13 rats, 50.66% ± 2.88%; FIP3 KD, n = 14 rats, 49.44% ± 2.48%, P >0.05; scrambled, n = 12 rats, 51.70% ± 2.90%, P >0.05. Test: control, n = 13 rats, 68.02% ± 3.12%; FIP3 KD, n = 14 rats, 50.74% ± 3.01%, P <0.01; scrambled, n = 12 rats, 64.38% ± 3.92%, P >0.05; one-way repeated-measures ANOVA vs control. K Schematic of the temporal order memory task. L FIP3 KD rats show deficits in the temporal order memory task. In the temporal order memory task, control and scrambled rats prefer the object explored at the early stage to that explored at the end, whereas the FIP3 KD rats have a comparable preference for the two objects. Control, n = 11 rats, 68.31% ± 3.20%; FIP3 KD, n = 15 rats, 52.14% ± 3.45%, P <0.01; scrambled, n = 11 rats, 68.07% ± 3.43%, P >0.05; one-way repeated-measures ANOVA vs control. The data are represented as the mean ± SEM, * P <0.05; ** P <0.01.

Article Snippet: The major primary antibodies were: MyoVa (Sigma, St. Louis, USA, HPA001356), GluN2A (Millipore, Burlington, USA, AB1555P), GluN1 (rabbit: Abclonal, Wuhan, China, A11699; mouse: Abcam, Cambridge, UK, ab134308), GluA1 (Millipore, MAB2263), GluN2B (Cell Signaling Technology, Boston, USA, 14544), Rab11 (Invitrogen, Carlsbad, USA, 71-5300), FIP3 (Rockland, Philadelphia, USA, 600-401-994), PSD95 (Millipore, MABN68), MyoVb (Sigma, HPA040902), CaMKII (Santa Cruz Biotechnology, Inc., Dallas, USA, sc-13141), GST (glutathione-S-transferase, Genscript, Nanjing, China, A00866-100).

Techniques: Expressing, Control