Article Figures & Data
Figures
- Fig. 1.
Construction and functional expression of VP22-GFP fusions. HVP22 and BVP22 gene homologs were amplified by PCR and subcloned into a GFP variant-encoding mammalian expression vector. (A) Nucleic acid and amino acid changes to the wild-type VP22 homolog (boldface) through the start codon for the fused GFP. (B) Western immunoblot of expressed protein from these constructs utilizing anti-GFP.
- Fig. 2.
Alignment of BVP22 and HVP22. BVP22 (259 amino acids) and HVP22 (301 amino acids) amino acid sequences were optimally aligned using a computer program (ALIGN) available from GeneStream. The results revealed only 28.7% homology. :, identity; · , similar acidity; global alignment score, 273.
- Fig. 3.
Intracellular localization of VP22 homologs. D17 cells were transiently transfected with BVP22 or HVP22 and analyzed by fluorescence microscopy. Scale bar, 2 μm.
- Fig. 4.
VP22 homolog filamentous pattern costains with microtubules. BVP22- or HVP22-transfected D17 cells were costained with α-tubulin antibody and analyzed by fluorescence microscopy. The same fields are shown in upper and lower panels using different filter sets. Scale bar, 2 μm.
- Fig. 5.
Colcemid treatment disrupted the filamentous cytoplasmic pattern of BVP22. Addition of Colcemid to BVP22-transfected D17 cells resulted in a diffuse cytoplasmic pattern (long arrow). However, the nuclear association pattern (short arrow) remained unchanged. Scale bar, 2 μm.
- Fig. 6.
Distinctions in the nuclear localization patterns of BVP22 and HVP22. Fluorescence microscopy of transiently transfected D17 cells revealed a marbled pattern of nuclear staining by BVP22 and a speckled nuclear staining by HVP22. Frequently, BVP22-stained nuclei would be attached by filaments. Scale bar, 2 μm.
- Fig. 7.
Nuclear membrane lysis of VP22 homolog-transfected D17 cells accentuates differences in nuclear protein fraction labeling. BVP22-transfected cultures have a halo appearance, whereas HVP22-transfected cultures have accentuated nucleolus labeling. Lysis of VP22-expressing cells (arrows; upper panels) resulted in nuclear labeling of the entire monolayer for both homologs. Scale bar, 2 μm.
- Fig. 8.
Both VP22 homologs bound chromatin during mitosis. Arrows, BVP22 or HVP22 bound to chromatin in metaphase (upper left panel) or telophase (all other panels) in transfected D17 cells. BVP22-transfected cells were viewed under both bright-field and fluorescence microscopy. HVP22-transfected cells were viewed under fluorescence microscopy only. Scale bar, 2 μm.
- Fig. 9.
Difference in the colocalizations of VP22 homologs and the mitosis marker phosphohistone H3 (phospho-H3). BVP22- or HVP22-transfected D17 cells were treated with Colcemid (4 h) to arrest cells in metaphase. Subsequently, transfects were stained with phosphohistone H3 antibody and colocalization was analyzed by fluorescence microscopy. The same fields are shown in upper and lower panels using filter sets for green (upper panels) and red (lower panels). Arrows, cells costaining for the VP22 homolog and phosphohistone H3. Scale bar, 2 μm.
- Fig. 10.
Difference in the colocalizations of VP22 homologs and BVP8. D17 cells were cotransfected with BVP22-GFP (BVP22) or HVP22-GFP (HVP22) and BVP8-BFP (BVP8) and analyzed using fluorescence microscopy. The same fields are shown in upper and lower panels using filter sets for green (upper panels) and blue (lower panels). Arrows, location of BVP8 spheres. BVP8 and BVP22 do not costain, whereas BVP8 costains with HVP22, indicating that BVP8 can partially sequester HVP22. Scale bar, 2 μm.
- Fig. 11.
BVP22 traffics to nontransfected cells in a monolayer. D17 or F17 cell monolayers were cotransfected with BVP22-GFP (BVP22) or HVP22-GFP (HVP22) and BFP (BFP[BVP22]; BFP[HVP22]). Nine random fields per coverslip of triplicate sets of nonfixed transfected cells were counted daily for numbers of blue fluorescing cells (BFP) and green fluorescing cells (BVP22 or HVP22). Data are expressed as means ± standard deviations and are representative of at least three experiments.
- Fig. 12.
Demonstration of VP22 homolog trafficking in live cells. D17 cell monolayers were cotransfected with BVP22-GFP (BVP22) or HVP22-GFP (HVP22) and BFP. After 3 days, cells were washed in PBS and analyzed by epifluorescence microscopy. Identical images are shown in the upper (green) and lower (blue) panels for BVP22-BFP- and HVP22-BFP-cotransfected cells using corresponding filter sets. Scale bar, 5 μm.
