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: Experimental cell research

Article Title: Distinct mechanisms enable inward or outward budding from late endosomes/multivesicular bodies

doi: 10.1016/j.yexcr.2018.08.027

Figure Lengend Snippet: Outwardly budded vesicles were isolated as described in Fig. 1B. A) Nanosight tracking analysis revealed the vesicles were 118+/−13.6 nm in size. B) Vesicles visualized using electron microscopy were approximately 100 nm in size. Scale bar = 100 nm. C) The amount of EGFR found in the budded vesicles is 6.5+/−1.5% of the total added into the reactions (c, lane 1). Outward budding is dependent on cytosolic components (lane 2 compared to lane 1) (p<.05; n=3). D) The intracellular epitope of the EGFR was cleaved from isolated outwardly budded vesicles by trypsin incubation. E) EGFR-containing outwardly budded vesicles were isolated from HeLa cells expressing a dominant negative Rab11 (Rab11S25N) construct containing a C-terminal myc epitope tag or a control vector (n=3). F) Vesicles derived from EGFRK721A expressing HeLa cells showed that 5+/−1.3% of total EGFRK721A is found in the budded vesicles (lane 1) and 19+/−4.8% of total EGFR K721A is protected from trypsin digestion (lane 2) (n=3). Data represents the mean +/− S.E.M. normalized to the control. *denotes p < 0.05).

Article Snippet: The PCMV-intron myc Rab11 S25N construct and the pcDNA3.1 control vector were purchased from Addgene.

Techniques: Isolation, Electron Microscopy, Incubation, Expressing, Dominant Negative Mutation, Construct, Control, Plasmid Preparation, Derivative Assay

Journal: Experimental cell research

Article Title: Distinct mechanisms enable inward or outward budding from late endosomes/multivesicular bodies

doi: 10.1016/j.yexcr.2018.08.027

Figure Lengend Snippet: Postnuclear supernatant was loaded onto a continuous 10–20% overnight opti-prep gradient. A) Fractions were collected and the refractive index was measured. B) Fractions were immunoblotted for endosomal markers (EEA1 for early endosomes, LAMP1/Rab7 for late endosomes, RAB11 for recycling endosomes, EGFR, and TfR). C) Trypsin resistance of early and late endosomal fractions. Top: Early endosomes. Left lane: EGFR signal without trypsin digestion Right lane: EGFR signal with trypsin digestion. Bottom: Late endosomes. Left lane: EGFR signal without trypsin digestion Right lane: EGFR signal with trypsin digestion. Late endosomal membranes contained more EGFR protected from trypsin cleavage compared to early endosomal membranes. D-H) Endosomes were visualized using cryo-electron microscopy and endosomal diameter was measured using IMOD. Endosomal distribution across fractions (D) and average endosome size (E) was determined. Fractions 3&4 contain significantly larger endosomes (p<0.0001; n=475). F) Endosomes in the range of 200–500nm (indicative of MVB size) in both fractions were then analyzed for presence or absence of internal vesicles with a diameter of 50 ± 10nm. In fractions 8&9, 20% of endosomes contained internal vesicles whereas 41.8% of endosomes in fractions 3&4 contained internal vesicles. Representative images captured using cryo-electron microscopy for fractions 8&9 (G) and fractions 3&4 (H) are shown. Arrowheads represent internal vesicles. Scale bar = 100 nm

Article Snippet: The PCMV-intron myc Rab11 S25N construct and the pcDNA3.1 control vector were purchased from Addgene.

Techniques: Refractive Index, Cryo-Electron Microscopy