Ubiquitin-Related Proteins Regulate Interaction of Vimentin Intermediate Filaments with the Plasma Membrane  Ai-Ling Wu, Jun Wang, Alexander Zheleznyak, Eric J Brown  Molecular Cell  Volume 4, Issue 4, Pages 619-625 (October 1999) DOI: 10.1016/S1097-2765(00)80212-9

Figure 1 The PLIC Family of IAP-Interacting Proteins (A) The four alternatively spliced cytoplasmic tails of IAP that arise from alternative splicing from a single promiscuous splice donor site (Reinhold et al. 1995). Cytoplasmic tails used in the assay are in bold. (B) Full-length PLIC-1 and PLIC-2 were ligated in-frame into the VP16 vector and cotransformed with IAP2 and IAP4 cytoplasmic tail baits or with baits consisting of integrin cytoplasmic tails (β2, αIIb, β3, and β3/β1 fusion) (Shattil et al. 1995) or nuclear lamin. Interaction was measured in suspension culture with a quantitative β-galactosidase assay; each value was obtained from the assays of four independent colonies. Inset: yeast expression of IAP2 and IAP4 cytoplasmic tail baits detected by antibody to the LexA fusion, showing approximately equal expression. No band is seen in lysate of untransformed L40 yeast cells. Antibody specific for IAP2 and IAP4 also showed that these were expressed in the appropriate yeast strains. (C) The carboxy-terminal UBA and amino-terminal UBQ domains of PLIC-1 and PLIC-2 compared to each other and to S. cerevisiae DSK-2 protein and to the F15C11.2 orf from C. elegans. Conservation of amino acids among the aligned sequences is shown by asterisks, indicating completely conserved positions, or by single crosses, indicating positions with conservation in three of the four proteins. In addition to identities, the following sets were considered to contain homologous amino acids: I, L, M, and V; K, R, and H; D, E, Q, and N; S and T; Y and F. Alignment was performed using the ClustalW program of the Network Proteins Sequence Analysis package at Pôle Bio-Informatique Lyonnais. (D) Expression of PLIC-1 and PLIC-2 mRNA. RNA from various E9 mouse organs was probed with PLIC-1 (upper) and PLIC-2 (lower) probes and compared to β-actin to assess loading. mRNAs for both PLIC-1 and PLIC-2 are ∼4 kb. Lanes 1–8 are in order: heart, brain, spleen, lung, liver, smooth muscle, kidney, and testis. Molecular Cell 1999 4, 619-625DOI: (10.1016/S1097-2765(00)80212-9)

Figure 2 IAP Interaction with PLICs (A) IAP purified from placenta (predominantly IAP2) (lane 1) was incubated with GST-PLIC-2 (lane 2), GST-PLIC-1 (lane 3), or GST-SHP-1 (lane 4). IAP bound after extensive washing was detected by SDS-PAGE and Western blotting. (B) OV10 cells expressing (+) or lacking (−) IAP were transfected with myc-tagged PLIC-1 and then incubated with beads coated with anti-β3 integrin mAb, and the integrin-associated complex purified as described in the Experimental Procedures. After separation of proteins on SDS-PAGE, PLIC-1 associated with the complex was detected by Western blotting with anti-myc. Presence of β3 integrin and IAP in the purified complex also was assessed, as was PLIC-1 in the total cell lysate (total). Equal amounts of αvβ3 integrin were purified whether or not IAP was also expressed. Total PLIC-1 expression was always at least as high in the cells without IAP as the cells with IAP. Beads coated with irrelevant mAb did not purify any αvβ3, IAP, or PLIC protein (data not shown). Molecular Cell 1999 4, 619-625DOI: (10.1016/S1097-2765(00)80212-9)

Figure 3 PLIC Expression Increases Cell Spreading (A) OV10 expressing (+) or lacking (−) IAP were transfected with myc-tagged PLICs or myc-tagged l-plastin. After 5 hr, cells were fixed and stained with anti-myc, and surface area of transfected (fluorescent) cells was measured. PLIC-transfected IAP+ cells had significantly greater area than PLIC-transfected IAP− cells or plastin-transfected IAP+ or − cells (p < 0.0001). (B) Jurkat cells stably transfected with PLIC-1 were allowed to adhere to surfaces coated with anti-IAP or anti-LFA-1, and cell surface area was measured at several time points. For both (A) and (B), at least 50 cells areas were measured in each experiment at each time point. Molecular Cell 1999 4, 619-625DOI: (10.1016/S1097-2765(00)80212-9)

Figure 4 PLIC Transfection Induces Vimentin Colocalization with IAP at Cell–Cell Borders A431 cells stably transfected with PLIC-1 (A–C and G–I) or empty vector (D–F) were stained for IAP (A, D, and G) together with vimentin (B and E) or tubulin (H). Overlays of the IAP and vimentin or tubulin images (C, F, and I) show that there is significant colocalization of IAP and vimentin at cell borders in PLIC-1-transfected cells, but much less colocalization of IAP and vimentin in control transfectants. There is little colocalization of IAP and tubulin at cell borders in PLIC-transfected cells or controls (data not shown). Molecular Cell 1999 4, 619-625DOI: (10.1016/S1097-2765(00)80212-9)

Figure 5 PLICs Associate with Membranes and Cytoskeleton (A) Membrane and cytoplasm of adherent, suspension, or cytochalasin D–treated NIH 3T3 cells were separated and probed for PLIC-2, tubulin, and paxillin. C, cytosol; M, membrane. Adh, adherent cells; Sus, suspension cells; Cyto D, cytochalasin D–treated cells. A similar distribution between membrane and cytosol was seen for PLIC-1 (data not shown). (B) Cytoskeleton (gel) was polymerized from NIH 3T3 cytosol in vitro and examined together with supernatant (sup) for distribution of PLIC-2, calreticulin, and vimentin. Molecular Cell 1999 4, 619-625DOI: (10.1016/S1097-2765(00)80212-9)